Cd4+ cd25+ t-cells activated to a specific antigen

ABSTRACT

The invention relates to a method of assessing whether a subject includes CD4+,CD25+ T cells that have been activated to a specific antigen. The method includes the steps of obtaining from the subject a sample of lymphocytes including CD4+,CD25+ T cells, incubating at least one portion of the sample of lymphocytes so as to promote, distinction of CD4+,CD25+ T cells that have been activated to the specific antigen from those CD4+,CD25+ T cells that have not been activated to the specific antigen, and thereafter determining whether CD4+,CD25+ T cells activated to the specific antigen are present in the sample. The invention further relates to methods of growing CD4+, CD25+ T cells that have been activated to a specific antigen in vitro and to methods of increasing tolerance in a subject using the CD4+, CD25+ T cells that have been grown in vitro.

FIELD OF THE INVENTION

The invention relates to a method of assessing whether a subjectcomprises CD4⁺,CD25⁺ T cells that have been activated to a specificantigen, a method for determining whether a subject is tolerant, orcapable of becoming tolerant, to an antigen, and to methods forincreasing or decreasing tolerance to an antigen in a subject.

BACKGROUND OF THE INVENTION

The immune system provides a mechanism to protect the body againstinfection by foreign entities such as infectious organisms or foreignantigens. Under normal conditions, the immune system is capable ofrecognising and eliciting an immune response against foreign entities,while largely ignoring host tissue. The ability of the immune system toignore the host's tissue is known as immune tolerance. Immune tolerancealso refers to a state where the immune system is adapted to ignoreantigens such as transplanted foreign tissues, infected tissues,allergens and malignant tissues.

Autoimmune disease occurs when T cells recognise and react to “self”molecules, that is, molecules produced by the cells of the host. Thisoccurs when specific self molecules interact with proteins on thesurface of T cells such that the T cells recognise the molecule asforeign and consequently elicit an immune response against the selfmolecule. In tissue transplantation, non-self major histocompatibilityantigen present on the foreign tissue contacts the surface of T cells,resulting in T-cell activation against the foreign antigen. Thisactivation ultimately results in allograft or xenograft rejection by theimmune system.

Present methods for preventing allograft rejection, or for treatingautoimmune disease, typically cause a general immunosuppression that isnot specific for a specific antigen or antigens. As a result, thesubject is rendered susceptible to infection from pathogenic andopportunistic organisms, and may be at an increased risk of malignancy.The more specific immunosuppressive drugs such as cyclosporin A,steroids, azathioprine, anti-T-cell antibodies, rapamycin, mycophenolatemofetil, desoxyspergualine and FK506, typically have undesirableside-effects, and typically require that the subject be administered thedrugs for life or at least extended periods of time, thereby placing thesubject at considerable risk of infection, cancer, and/or otherconditions due to long term effects of the treatment. It would thereforebe advantageous to provide a method of inducing tolerance to a specificantigen in a subject. Such a method could be used to suppress the immuneresponse to “self” molecules in a subject having an autoimmune disease,or to suppress the immune response to transplant tissue by inducingtolerance to antigens present on the transplant tissue.

Disease conditions may also result from, or be exacerbated by, thedevelopment of immune tolerance to a specific antigen. For example,diseases such as cancer and chronic infections may result from, orprogress because of, the development of immune tolerance to tumour orother antigens present on the malignant or pre-malignant cells orantigens of infectious agents expressed by infected cells. It wouldtherefore also be desirable to provide a method of reducing or breakingtolerance to a specific antigen in a subject suffering from diseaseresulting from or exacerbated by the development of immune tolerance toa specific antigen.

It would further be desirable to provide a method for assessing whethera subject is capable of becoming tolerant to a specific antigen. Forexample, treatment of patients with immunosuppressive drugs such ascyclosporin A can in some cases lead to tolerance or partial toleranceto specific antigens such as self molecules or alloantigens. If theonset of tolerance could be detected in a patient on suchimmunosuppressive drug therapy, or in other circumstances, an assessmentcould be made as to whether the patient still required high levels ofimmunosuppressive drugs, or whether they had developed sufficienttolerance to a specific antigen to permit the dose of immunosuppressivedrugs to be reduced or eliminated.

SUMMARY OF THE INVENTION

CD4⁺ T cells are a subset of lymphocytes that are central to inducing animmune response in a human or animal body. CD4⁺,CD25⁺ T cells are asubpopulation of CD⁺ T cells which represent approximately 1% to 10% ofthe total CD⁺ T cell population in a human or animal body.

CD4⁺, CD25⁺ T cells activated to an antigen are capable of imparting tocells of the immune system, including CD4⁺, CD25⁻ T cell populations andCD8⁺ T cells, tolerance to that antigen.

Activated CD4⁺,CD25⁺ T cells are formed when naïve CD4⁺,CD25⁺ T cellscontact an antigen in the presence of interleukin-2 (IL-2) and/orinterleukin-4 (IL-4). The activated CD4⁺,CD25⁺ T cells initiallyproliferate following activation. However, the inventors have found thatactivated CD4⁺,CD25⁴⁺ T cells die, or are not capable of furtherproliferation after an initial proliferation following activation,unless exposed to specific cytokines.

The inventors have found that naïve CD4⁺,CD25⁺ T cells do not expressreceptors for IL-5, IL-12 or IFN-γ. However, the inventors have foundthat when naïve CD4⁺,CD25⁺ T cells are activated by contacting the naïveCD4⁺,CD25⁺ T cells with an antigen and IL-2, the CD4⁺,CD25⁺ T cellsexpress the IFN-γ receptor and the IL-12 β2 receptor. The inventors havefound that when naïve CD4⁺,CD25⁺ T cells are activated by contacting thenaïve CD4⁴⁺,CD25⁺ T cells with an antigen and IL-4, the CD4⁺,CD25⁺ Tcells express the IL-5 α-receptor, and expression of IFN-γ mRNAincreases. The inventors have found that the cytokines interleukin-5(IL-5), interleukin-12 (IL-12), interleukin-23 (IL-23) and interferon-γ(IFN-γ) are capable of prolonging the survival, and supportingproliferation, of activated CD4⁺,CD25⁺ T cells.

Based on their findings, the inventors have developed methods fordetermining whether a subject comprises CD4⁺,CD25⁺ T cells that havebeen activated to a specific antigen, methods for determining whether asubject is tolerant, or capable of becoming tolerant, to a specificantigen, methods for growing CD4⁺,CD25⁺ T cells activated to a specificantigen, and methods for inducing and reducing tolerance to a specificantigen.

In a first aspect, the invention provides a method of assessing whethera subject comprises CD4⁺,CD25⁺ T cells that have been activated to aspecific antigen, comprising the steps of:

-   -   (a) obtaining from the subject a sample of lymphocytes        comprising CD4⁺,CD25⁺ T cells;    -   (b) incubating at least one portion of the sample of lymphocytes        so as to promote distinction of CD4⁺,CD25⁺ T cells that have        been activated to the specific antigen from CD4⁺,CD25⁺ T cells        that have not been activated to the specific antigen;    -   (c) thereafter determining whether CD4⁴⁺,CD25⁺ T cells that have        been activated to the specific antigen are present in the        sample.

Typically, step (b) of the method comprises incubating the at least oneportion of the sample under conditions that favour the survival and/orproliferation of CD4⁺,CD25⁺ T cells that have been activated to thespecific antigen in preference to other CD4⁺,CD25⁺ T cells, orincubating the at least one portion of the sample under conditions thatfavour the survival and/or proliferation of CD4⁺,CD25⁺ T cells that havenot been activated to the specific antigen in preference to CD4⁺,CD25⁺ Tcells that have been activated to the specific antigen.

In some embodiments, the at least one portion of the sample is incubatedin the presence of the specific antigen and one or more cytokinescapable of prolonging survival and/or supporting proliferation ofactivated CD4⁺,CD25⁺ T cells. The incubation of the at least one portionof the sample under these conditions favours the survival and/orproliferation of CD4⁺,CD25⁺ T cells that have been activated to thespecific antigen. The one or more cytokines capable of prolongingsurvival or supporting proliferation of activated CD4⁺,CD25⁺ T cells aretypically cytokines selected from the group consisting of IL-5, IL-12,IL-23 and IFN-γ, biologically active fragments thereof, and functionallyequivalent molecules thereof. In these embodiments, it can be determinedthat CD4⁺,CD25⁺ T cells that have been activated to the specific antigenare present in the sample if:

-   -   (a) proliferation of CD4⁺,CD25⁺ T cells is detected;    -   (b) in samples comprising CD4⁺,CD25⁺ and CD4⁺,CD25⁻ T cells,        there is a reduction of CD4⁺,CD25⁻ T cell proliferation in        response to the specific antigen; or    -   (c) molecules are detected which indicate the presence of        CD4⁺,CD25⁺ T cells that have been activated to the specific        antigen.

In some other embodiments, the at least one portion of the sample isincubated in the presence of the specific antigen and in the absence ofcytokines capable of stimulating activation of CD4⁺,CD25⁺ T cells or ofprolonging survival or supporting proliferation of activated CD4⁺,CD25⁺T cells. In these embodiments, it can be determined that CD4⁺,CD25⁺ Tcells that have been activated to the specific antigen are present inthe sample if:

-   -   (a) there is reduced proliferation of CD4⁺,CD25⁺ T cells;    -   (b) in samples comprising CD4⁺,CD25⁺ and CD4⁺,CD25⁻ T cells,        there is increased proliferation of CD4⁺,CD25⁻ T cell in        response to the specific antigen.

In the absence of cytokines capable of prolonging survival or supportingproliferation of activated CD4⁺,CD25⁺ T cells, CD4⁺,CD25⁺ T cellsactivated to a specific antigen will not proliferate following contactwith the specific antigen, and typically will not survive, or willexhibit reduced proliferation relative to CD4⁺,CD25⁺ T cells that havenot been activated to the specific antigen. Accordingly, when the atleast one portion of the sample is incubated in the presence of thespecific antigen and in the absence of cytokines capable of stimulatingactivation of CD4⁺,CD25⁺ T cells or of prolonging survival or supportingproliferation of activated CD4⁺,CD25⁺ T cells, a reduction in survivaland/or proliferation of CD4⁺,CD25⁺ T cells will indicate the presence ofCD4⁺,CD25⁺ T cells activated to the specific antigen in the sample. In asample comprising CD4⁺,CD25⁺ and CD4⁺,CD25⁻ T cells, a reduction insurvival and/or proliferation of CD4⁺,CD25⁺ T cells will result in anincrease in proliferation of the CD4⁺,CD25⁻ T cells. This is because areduction in survival and/or proliferation of CD4⁺,CD25⁺ T cells in thesample will mean these cells are no longer able to suppress theproliferation of CD4⁺,CD25⁻ T cells in response to the specific antigen.On the other hand, naïve CD4⁺,CD25⁺ T cells and CD4⁺,CD25⁺ T cells thathave been activated to other antigens will survive when incubated underthese conditions, and will be able to suppress proliferation ofCD4⁺,CD25⁺ T cells.

The presence of CD4⁺,CD25⁺ T cells activated to a specific antigen inthe sample indicates that the subject is tolerant, or capable ofbecoming tolerant, to the specific antigen.

In a second aspect, the invention provides a method of determiningwhether a subject is tolerant, or capable of becoming tolerant, to aspecific antigen, comprising the steps of:

-   -   (a) obtaining from the subject a sample of lymphocytes        comprising CD4⁺,CD25⁺ T cells;    -   (b) contacting at least one portion of the sample of lymphocytes        with the specific antigen;    -   (c) incubating the at least one portion of the sample of        lymphocytes in the absence of cytokines capable of stimulating        activation of CD4⁺,CD25⁺ T cells or of prolonging survival or        supporting proliferation of activated CD4⁺,CD25⁺ T cells, or in        the presence of at least one cytokine selected from the group        consisting of IL-5, IL-12, IL-23 and IFN-γ, or a biologically        active fragment thereof, or a functionally equivalent molecule        thereof;    -   (d) thereafter determining whether CD4⁺,CD25⁺ T cells that have        been activated to the specific antigen are present in the        sample.

The presence of CD4⁺,CD25⁺ T cells activated to the specific antigen inthe sample may be determined by detecting:

-   -   (i) no or low proliferation of CD4⁺,CD25⁺ T cells when the at        least one portion of the sample of lymphocytes is contacted with        the specific antigen and incubated in the absence of cytokines        capable of stimulating activation of CD4⁺,CD25⁺ T cells or of        prolonging survival and/or supporting proliferation of activated        CD4⁺,CD25⁺ T cells; or    -   (ii) proliferation of CD4⁺,CD25⁺ T cells when the at least one        portion of the sample of lymphocytes is contacted with the        specific antigen and incubated in the presence of at least one        cytokine selected from the group consisting of IL-5, IL-12,        IL-23 and IFN-γ, or a biologically active fragment thereof, or a        functionally equivalent molecule thereof; or    -   (iii) molecules which indicate the presence of activated        CD4⁺,CD25⁺ T cells when the at least one portion of the sample        of lymphocytes is contacted with the specific antigen and        incubated in the presence of at least one cytokine selected from        the group consisting of IL-5, IL-12, IL-23 and IFN-γ, or a        biologically active fragment thereof, or a functionally        equivalent molecule thereof; or    -   (iv) inhibition of proliferation of CD4⁺,CD25⁻ T cells when the        at least one portion of the sample comprises CD4⁺,CD25⁺ T cells        and CD4⁺ CD25⁻ T cells, and the at least one portion of the        sample of lymphocytes is contacted with the specific antigen and        incubated in the presence of at least one cytokine selected from        the group consisting of IL-5, IL-12, IL-23 and IFN-γ, or a        biologically active fragment thereof, or a functionally        equivalent molecule thereof; and/or    -   (v) stimulation of proliferation of CD4⁺,CD25⁻ T cells when the        at least one portion of the sample comprises CD4⁺,CD25⁺ T cells        and CD4⁺,CD25⁻ T cells, and the at least one portion of the        sample of lymphocytes is contacted with the specific antigen and        incubated in the absence of cytokines capable of stimulating        proliferation of CD4⁺,CD25¹⁻ T cells, or prolonging survival or        supporting proliferation of activated CD4⁺,CD25⁺ T cells.

Molecules which indicate the presence of activated CD4⁺,CD25⁺ T cellsmay be metabolic products that are signals of activation, or markers ofactivation such as expression of the IL-5 receptor, the IL-12 receptor,or the IFN-γ receptor.

Typically, the IL-12 is IL-12p70.

In one embodiment, the at least one portion of the sample of lymphocytesis incubated in the absence of cytokines capable of stimulatingactivation of CD4⁺,CD25⁺ T cells or of prolonging survival orstimulating proliferation of activated CD4⁺,CD25⁺ T cells and in thepresence of anti-IL-2 and/or anti-IL-4 antibodies. In this embodiment,the presence of activated CD4⁺,CD25⁺ T cells in the sample may bedetermined by detecting no or low proliferation of CD4⁺,CD25⁺ T cells.

In one embodiment, the at least one portion of the sample of lymphocytesis incubated in the presence of at least one cytokine selected from thegroup consisting of IL-5, IL-12, IL-23 and IFN-γ, or a biologicallyactive fragment thereof, or a functionally equivalent molecule thereof,and in the presence of anti-IL-2 and/or anti-IL-4 antibody. In thisembodiment, the presence of activated CD4⁺,CD25⁺ T cells in the samplemay be determined by detecting proliferation of CD4⁺,CD25⁺ T cells.

The sample of lymphocytes may be any population of lymphocytes obtainedfrom the subject which contains CD4⁺,CD25⁺ T cells. In one embodiment,the sample of lymphocytes is isolated CD4⁺,CD25⁺ T cells. In anotherembodiment, the sample is isolated CD4⁺ T cells. In yet anotherembodiment, the sample is a mixed lymphocyte population. Typically, themixed lymphocyte population is unfractionated lymphocytes. In yetanother embodiment, the sample may be an admixed T cell populationobtained by mixing isolated CD4⁺,CD25⁺ T cells and isolated CD4⁴⁺,CD24⁻T cells obtained from the subject.

In one embodiment, at least one portion of the sample of lymphocytes isincubated with an antibody which reduces proliferation of CD4⁺,CD25⁻lymphocytes. Antibodies which reduce the proliferation of CD4⁺,CD25⁻ Tcells include anti-CD3, anti-CD45RB/RO or any other antibody whichspecifically binds to CD4⁺,CD25⁻ T cells.

The lymphocytes may be contacted with the specific antigen in any mannerwhich presents the specific antigen to the lymphocyte in a form whichwill permit the lymphocyte to recognise the specific antigen. The atleast one portion of the sample of lymphocytes may be contacted with thespecific antigen prior to, or simultaneously with, incubating at leastone portion of the sample of lymphocytes in the absence of cytokinescapable of stimulating activation of CD4⁺,CD25⁺ T cells or of prolongingsurvival or supporting proliferation of activated CD4⁺,CD25⁺ T cells, orin the presence of at least one cytokine selected from the groupconsisting of IL-5, IL-12, IL-23 and IFN-γ, or a biologically activefragment thereof, or a functionally equivalent molecule thereof. In someembodiments the at least one portion of the sample is contacted with thespecific antigen by incubating the at least one portion of the samplewith the specific antigen.

The specific antigen may be an antigen located on the surface of anantigen presenting cell. In such a case, the specific antigen may beassociated with a class II major histocompatability molecule on thesurface of the antigen presenting cell. The antigen presenting cell maybe any cell that expresses an antigen presenting molecule (typicallyclass II MHC) and, typically, ligands required to facilitate activationof the CD4⁺,CD25⁺ T cells. Examples of ligands include ICAM1, ICAM2,LFA3, ligands for CD28 and CTLA-4 or any activation ligands. Examples ofantigen presenting cells include dendritic cells, phagocytes,B-lymphocytes, Langerhans cells or unfractionated lymphocytes in whichthe proliferation of the stimulator cells is impaired (for example, byirradiation or mitomycin C treatment).

Alternatively, the at least one portion of the sample of lymphocytes maybe contacted with the specific antigen in a synthetic antigen presentingsystem such as that described in, for example, U.S. Pat. No. 6,828,150or 6,787,154.

Where the at least one portion of the sample of lymphocytes is incubatedin the presence of at least one cytokine selected from the groupconsisting of IL-5, IL-12, IL-23 and IFN-γ, or a biologically activefragment thereof, or a functionally equivalent molecule thereof, the atleast one portion of the sample of lymphocytes may be incubated in thepresence of the at least one cytokine simultaneous with, or subsequentto, contacting the at least one portion of the sample of lymphocyteswith the specific antigen.

Typically, the at least one portion of the sample of lymphocytes isincubated in medium. The medium is typically substantially free ofexogenous antigens which may cause activation of lymphocytes, other thanthe specific antigen. In other words, the levels of exogenous antigenpresent in the medium are sufficiently low, or absent, such that CD4⁺ Tcells do not activate to exogenous antigens (other than the specificantigen) in the medium.

When the at least one portion of the sample of lymphocytes is incubatedin the presence of at least one cytokine selected from the groupconsisting of IL-5, IL-12, IL-23 and IFN-γ, or a biologically activefragment thereof, or a functionally equivalent molecule thereof, the atleast one cytokine selected from the group consisting of IL-5, IL-12,IL-23′ and IFN-γ, or a biologically active fragment thereof, or afunctionally equivalent molecule thereof, is typically added to themedium from an exogenous source. The at least one cytokine selected fromthe group consisting of IL-5, IL-12, IL-23 and IFN-γ, or a biologicallyactive fragment thereof, or a functionally equivalent molecule thereof,may be purified proteins, recombinant or otherwise. Alternatively, theat least one portion of the sample of lymphocytes may be incubated inthe presence of cells which express the at least one cytokine selectedfrom the group consisting of IL-5, IL-12, IL-23 and IFN-γ, or abiologically active fragment thereof, or a functionally equivalentmolecule thereof. The medium may comprise growth factors, nutrientsand/or buffers.

Typically, when the sample of lymphocytes is incubated in the absence ofcytokines capable of stimulating activation of CD4⁺,CD25⁻ T cells or ofprolonging survival or stimulating proliferation of activated CD4⁺,CD25⁺T cells, the sample is incubated in medium which is substantially freeof cytokines.

Proliferation of the CD4⁺,CD25⁺ T cells and CD4⁺,CD25⁻ T cells may bedetermined by any methods known in the art. For example, proliferationof the CD4⁺,CD25⁺ T cells may be determined by determining the number ofCD4⁺,CD25⁺ T cells, or determining the proportion of CD4⁺,CD25⁺ T cellsthat are proliferating.

The number of CD4⁺,CD25⁺ T cells or CD4⁺,CD25⁻ T cells may be determinedusing any methods known in the art for measuring cell numbers. Suchmethods include flow cytometry, immunofluorescent microscopy, etc.

The proportion of proliferating CD4⁺,CD25⁺ T cells and/or CD4⁺,CD25⁻ Tcells may be determined by methods such as rate of incorporation ofradiolabels such as [H³]-thymidine or incorporation of fluorescent dyessuch as ethidium bromide, acridine orange etc.

The specific antigen may be any antigen. The specific antigen may be anautoantigen of the subject and the subject has an autoimmune disease orcondition.

The specific antigen may be an alloantigen, for example, an antigen ofallograft tissue following, or prior to, an allograft to the subject, orin other words, a transplant of tissue to the subject from anothersubject of the same species.

The specific antigen may be a xenoantigen, for example, an antigen ofxenograft tissue following, or prior to, a xenograft to the subject, orin other words, a transplant of tissue to the subject from a speciesdifferent to that of the subject.

The specific antigen may be an allergen or part of an allergen thatinduces the allergic response.

The specific antigen may be a tumour antigen. An example of a tumourantigen is a neoantigen, a tumour cell or a pre-malignant cell.

The specific antigen may be an antigen from an infectious agent.

The specific antigen may be a single specific antigen, or a plurality ofspecific antigens.

The specific antigen may be a cell transfected with an agent such as atherapeutic agent. In such a case, the cell is typically autologous tothe subject. Typically, the cell transfected with an agent is a stemcell.

In a third aspect, the invention provides a method of growing in vitroCD4⁺,CD25⁺ T cells activated to a specific antigen, comprising the stepof culturing CD4⁺,CD25⁺ T cells activated to the specific antigen invitro in the presence of at least one cytokine selected from the groupconsisting of IL-5, IL-12, IL-23 and IFN-γ, or a biologically activefragment thereof, or a functionally equivalent molecule thereof.

In one embodiment, the method comprises the further step of contactingthe CD4⁺,CD25⁺ T cells activated to the specific antigen with thespecific antigen in vitro. The activated CD4⁺,CD25⁺ T cells may becontacted with the specific antigen in vitro prior to, or simultaneouswith, culturing the T cells in the presence of the at least one cytokineselected from the group consisting of IL-5, IL-12, IL-23 and IFN-γ, or abiologically active fragment thereof, or a functionally equivalentmolecule thereof. In some embodiments, the activated CD4⁺,CD25⁺ T cellsare cultured in the presence of the at least one cytokine selected fromthe group selected from IL-5, IL-12, IL-23 and IFN-γ, or a biologicallyactive fragment thereof, or a functionally equivalent molecule thereof,after contacting the activated T cells with the specific antigen.

Typically, the activated CD4⁺,CD25⁺ T cells are cultured in the absenceof conconavalin A and/or 1-o-methyl α-mannopyranoside.

Typically, the IL-12 is IL-12p70.

In a fourth aspect, the invention provides a method of increasingtolerance to a specific antigen in a subject in need thereof, comprisingadministering to the subject an effective amount of CD4⁺,CD25⁺ T cellsactivated to the specific antigen grown in vitro by culturing theactivated CD4⁺,CD25⁺ T cells in the presence of at least one cytokineselected from the group consisting of IL-5, IL-12, IL-23 and IFN-γ, or abiologically active fragment thereof, or a functionally equivalentmolecule thereof.

In one embodiment, the method comprises the further step of contactingthe CD4⁺,CD25⁺ T cells activated to the specific antigen with thespecific antigen in vitro.

The CD4⁺,CD25⁺ T cells activated to a specific antigen may be contactedwith the specific antigen in vitro prior to, or simultaneous with,culturing the activated T cells in the presence of the at least onecytokine selected from the group consisting of IL-5, IL-12, IL-23 andIFN-γ, or a biologically active fragment thereof, or a functionallyequivalent molecule thereof. In some embodiments, the T cells arecultured in the presence of the at least one cytokine selected from thegroup consisting of IL-5, IL-12, IL-23 and IFN-γ, or a biologicallyactive fragment thereof, or a functionally equivalent molecule thereofafter contacting the activated T cells with the specific antigen.

Typically, the CD4⁺,CD25⁺ T cells activated to a specific antigen arecultured in vitro in the absence of conconavalin A and/or 1-o-methylα-mannopyranoside.

Typically, the IL-12 is IL-12p70.

In one embodiment, the CD4⁺,CD25⁺ T cells activated to a specificantigen are cultured in vitro in the absence of IL-2 and IL-4.

Without wishing to be bound by theory, the inventors believe thattolerance to a specific antigen may be increased by increasing the ratioof CD4⁺,CD25⁺ T cells activated to the specific antigen relative to theCD4⁺,CD25⁻ T cells in a subject. Administering CD4⁺,CD25⁺ T cellsactivated to the specific antigen, increases the ratio of CD4⁺,CD25⁺ Tcells activated to the specific antigen to CD4⁺,CD25⁻ T cells in thesubject.

The ratio of CD4⁺,CD25⁺ T cells activated to the specific antigen toCD4⁺,CD25⁻ T cells in a subject may be further increased by reducing thenumber of CD4⁺ T cells in the subject prior to administering theCD4⁺,CD25⁺ T cells activated to the specific antigen. The CD4⁺ T cellsmay be reduced in number by any methods known in the art.

Lymphocytes, including CD4⁺ T cells, may be reduced by irradiation. TheCD4⁺ lymphocytes may be reduced by administering to the subject one ormore antibodies which specifically bind to CD4⁺ T cells, typically toCD4⁺,CD25⁻ T cells. Suitable antibodies include one or more antibodiesselected from the group consisting of anti-CD3, anti-CD4,anti-CD45RB/R4, anti-lymphocyte globulin or anti-thymocyte globulin. Inembodiments where the CD4⁺ T cells are reduced by administeringantibodies that specifically bind to the CD4⁺ T cells or lymphocytes,the antibodies may be subsequently removed or inactivated. Methods forremoval or inactivation of antibodies include administration ofanti-idiotype antibodies, soluble CD4 ligand, antibodies against thetreating antibody or any other technique that removes or neutralizes thetreating antibody.

In one embodiment, the CD4⁺,CD25⁺ T cells activated to the specificantigen are derived from naïve T cells that are activated in vitro. Thenaïve T cells may be activated in vitro by contacting naïve CD4⁺,CD25⁺ Tcells with the specific antigen in vitro, and culturing the T cells inthe presence of one or more cytokines capable of supporting activationof naïve CD4⁺,CD25⁺ T cells. Typically, the cytokine capable ofsupporting activation of the naïve CD4⁺,CD25⁺ T cells is IL-2, abiologically active fragment thereof, or a functionally equivalentmolecule thereof, or IL-4, a biologically active fragment thereof, or afunctionally equivalent molecule thereof.

The naïve CD4⁺,CD25⁺ T cells may be activated in vitro by:

-   -   (a) contacting the naïve CD4⁺,CD25⁺ T cells with the specific        antigen; and    -   (b) culturing the CD4⁺,CD25⁺ T cell in the presence of IL-2, a        biologically active fragment thereof, or functionally equivalent        molecule thereof.

Typically, CD4⁺,CD25⁺ T cells that have been activated in the presenceof IL-2, a biologically active fragment thereof, or functionallyequivalent molecule thereof, are simultaneously, or subsequently,cultured in the presence of IL-12, a biologically active fragmentthereof, or a functionally equivalent molecule thereof, and/or IFN-γ, abiologically active fragment thereof, or a functionally equivalentmolecule thereof. Naive CD4⁺,CD25⁺ T cells may alternatively activatedin vitro by:

-   -   (a) contacting the naïve CD4⁺,CD25⁺ T cells with the specific        antigen; and    -   (b) culturing the CD4⁺,CD25⁺ T cell in the presence of IL-4, a        biologically active fragment thereof, or functionally equivalent        molecule thereof.

In some embodiments, the CD4⁺,CD25⁺ T cells that have been activated inthe presence of IL-4, a biologically active fragment thereof, orfunctionally equivalent molecule thereof, are simultaneously, orsubsequently, cultured in the presence of IL-5 or IL-13, a biologicallyactive fragment thereof, or a functionally equivalent molecule thereof.In some embodiments, naïve CD4⁺,CD25⁺ T cells that have been activatedin the presence of IL-4, a biologically active fragment thereof, or afunctionally equivalent molecule thereof, may be cultured in thepresence of IL-5, a biologically active fragment thereof, or afunctionally equivalent molecule thereof, and IL-13, a biologicallyactive fragment thereof, or a functionally equivalent molecule thereof.

Naive CD4⁺,CD25⁺ T cells may be activated in vitro by:

-   -   (a) contacting the naïve CD4⁺,CD25⁺ T cells with the specific        antigen; and    -   (b) culturing the CD4⁺,CD25⁺ T cells in the presence of IL-2, a        biologically active fragment thereof, or a functionally        equivalent molecule thereof, and IL-4, a biologically active        fragment thereof, or functionally equivalent molecule thereof.

Typically, the CD4⁺,CD25⁺ T cells that have been activated in thepresence of IL-2, a biologically active fragment thereof, or afunctionally equivalent molecule thereof, and IL-4, a biologicallyactive fragment thereof, or a functionally equivalent molecule thereof,are simultaneously, or subsequently, cultured in the presence of one ormore of the cytokines selected from the group consisting of IL-5, IL-12,IL-23 and IFN-γ, or a biologically active fragment thereof, or afunctionally equivalent molecule thereof. The CD4⁺,CD25⁺ T cells thathave been activated in the presence of IL-2, a biologically activefragment thereof, or a functionally equivalent molecule thereof, andIL-4, a biologically active fragment thereof, or a functionallyequivalent molecule thereof, may further be cultured in the presence ofIL-13, a biologically active fragment thereof, or a functionallyequivalent molecule thereof.

In another embodiment, the CD4⁺,CD25⁺ T cells may be activated to thespecific antigen in vivo prior to culturing in vitro. For example,CD4⁺,CD25⁺ T cells may be isolated from the subject already activated tothe specific antigen. It is envisaged that the CD4⁺,CD25⁺ T cellsactivated to a specific antigen from the subject would typically beactivated following contact with specific antigen and exposure to IL-2and/or IL-4 in the subject, or in other words, in vivo.

It is also envisaged that a sample of CD4⁺,CD25⁺ T cells from a subjectmay contain naïve CD4⁺,CD25⁺ T cells and CD4⁺,CD25⁺ T cells which havebeen activated to the specific antigen. These cells may be grown bycontacting the naïve CD4⁺,CD25⁺ T cells and CD4⁺,CD25⁺ T cells activatedto the specific antigen with the specific antigen in vitro, andculturing the naïve CD4⁺,CD25⁺ T cells and CD4⁺,CD25⁺ T cells activatedto the specific antigen in the presence of IL-2, a biologically activefragment thereof, or a functionally equivalent molecule thereof, and/orIL-4, a biologically active fragment thereof, or a functionallyequivalent molecule thereof, and at least one cytokine selected from thegroup consisting of IL-5, IL-12, IL-23 and IFN-γ, or a biologicallyactive fragment thereof, or a functionally equivalent molecule thereof.In some embodiments, the CD4⁺,CD25⁺ T cells may further be cultured inthe presence of IL-13, a biologically active fragment thereof, or afunctionally equivalent molecule thereof.

In the method of the third or fourth aspect of the present invention,the CD4⁺,CD25⁺ T cells activated to the specific antigen may be culturedin any medium which comprises at least one cytokine selected from thegroup consisting of IL-5, IL-12, IL-23 and IFN-γ, or a biologicallyactive fragment thereof, or a functionally equivalent molecule thereof.The medium is typically free of conconavalin A and/or 1-o-methylα-mannopyranoside. The medium may contain, in addition to at least onecytokine selected from the group consisting of IL-5, IL-12, IL-23 andIFN-γ, or a biologically active fragment thereof, or a functionallyequivalent molecule thereof, one or more cytokines selected from thegroup consisting of IL-2, IL-4, biologically active fragments thereof,and functionally equivalent molecules thereof. The medium may contain,in addition to at least one cytokine selected from the group consistingof IL-5, IL-12, IL-23 and IFN-γ, or a biologically active fragmentthereof, or a functionally equivalent molecule thereof, one or moreantibodies which are directed against CD4⁺,CD25⁻ T cells. Examples ofsuch antibodies include anti-CD3, anti-CD45RB/RO. The medium may inaddition contain IL-13, a biologically active fragment thereof, or afunctionally equivalent molecule thereof.

The CD4⁺,CD25⁺ T cells activated to the specific antigen are typicallycultured in the presence of at least one cytokine selected from thegroup consisting of IL-5, IL-12, IL-23 and IFN-γ, or a biologicallyactive fragment thereof, or a functionally equivalent molecule thereof,wherein the at least one cytokine selected from the group consisting ofIL-5, IL-12, IL-23 and IFN-γ, or a biologically active fragment thereof,or a functionally equivalent molecule thereof has been added exogenouslyto the medium in which the CD4⁺,CD25⁺ T cells are cultured. The at leastone cytokines selected from the group consisting of IL-5, IL-12, IL-23and IFN-γ, or a biologically active fragment thereof, or a functionallyequivalent molecule thereof, may be constituents of a compositioncomprising other cytokines, growth factors, nutrients and/or buffers.The at least one cytokines selected from the group consisting of IL-5,IL-12, IL-23 and IFN-γ, or a biologically active fragment thereof, or afunctionally equivalent molecule thereof may be purified proteins,recombinant or otherwise, which are added directly to the medium inwhich the lymphocytes are cultured. Alternatively, the CD4⁺,CD25⁺ Tcells activated to the specific antigen grown in vitro may be culturedin the presence of cells which overexpress the at least one cytokineselected from the group consisting of IL-5, IL-12, IL-23 and IFN-γ, or abiologically active fragment thereof, or a functionally equivalentmolecule thereof, and which are co-cultured with the lymphocytes.

The CD4⁺,CD25⁺ T cells activated to the specific antigen may becontacted with the specific antigen in vitro in any manner which permitsthe lymphocyte to recognise the antigen. Typically, the CD4⁺,CD25⁺ Tcells activated to the specific antigen are contacted with the specificantigen located on the surface of an antigen presenting cell. Thespecific antigen may be displayed on the surface of the antigenpresenting cell associated with a class II major histocompatibilitymolecule. The antigen presenting cell may be any cell that expresses anantigen presenting molecule (typically class II MHC) and, typically,ligands required to facilitate activation of the CD4⁺,CD25⁺ T cells.Examples of ligands include ICAM1, ICAM2, LFA3, ligands for CD28 andCTLA-4 or any activation ligands. Examples of antigen presenting cellsinclude dendritic cells, phagocytes, B-lymphocytes, Langerhans cells orunfractionated lymphocytes in which the proliferation of the stimulatorcells is impaired (for example, by irradiation or mitomycin Ctreatment).

Alternatively, the CD4⁺,CD25⁺ T cell activated with the specific antigenmay be contacted with the specific antigen in a synthetic antigenpresenting system such as that described in, for example, U.S. Pat. No.6,828,150 or 6,787,154.

In one embodiment, the method of increasing tolerance to a specificantigen in a subject in need thereof comprises the further step ofadministering an effective amount of at least one cytokine selected fromthe group consisting of IL-5, IL-12, IL-23 and IFN-γ, or a biologicallyactive fragment thereof, or a functionally equivalent molecule thereof,simultaneously with, or subsequent to, administering the CD4⁺,CD25⁺ Tcells activated to the specific antigen. The method of increasingtolerance to a specific antigen in a subject may further compriseadministering an effective amount of IL-13, a biologically activefragment thereof, or a functionally equivalent molecule thereof.

In a fifth aspect, the invention provides a method of reducing toleranceto a specific antigen in a subject by reducing or eliminating theactivity of one or more cytokines selected from the group consisting ofIL-5, IL-12, IL-23 and IFN-γ, or a biologically active fragment thereof,or a functionally equivalent molecule thereof, in the subject to therebydecrease the ratio of CD4⁺,CD25⁺ T cells activated to the specificantigen relative to CD4⁺,CD25⁻ T cells.

The ratio of CD4⁺,CD25⁺ T cells activated to the specific antigen toCD4⁺,CD25⁻ T cells may be further decreased by administering to thesubject an effective amount of an agent which increases production ofCD4⁺,CD25⁻ T cells, and depletes CD4⁺,CD25⁺ T cells. Typically, theagent is IL-2, a biologically active fragment thereof, or a functionallyequivalent molecule thereof, and/or IL-4, a biologically active fragmentthereof, or a functionally equivalent molecule thereof. The agent may bean anti-CD25 antibody, typically an anti-CD25 monoclonal antibody.

The activity of IL-5, IL-12, IL-23 or IFN-γ may be reduced or eliminatedby any means for reducing or eliminating the biological activity of acytokine in a subject. For example, the activity of IL-5, IL-12, IL-23or IFN-γ may be reduced or eliminated by administering an effectiveamount of an antagonist of IL-5, IL-12, IL-23 and/or IFN-γ. Theantagonist may be any molecule which reduces or eliminates the activityof IL-5, IL-12, IL-23 and/or IFN-γ. Examples of suitable antagonistsinclude antibody receptor molecules, antisense molecules, IRNA or siRNAmolecules, or any other molecules which reduce or eliminate thebiological activity of IL-5, IL-12, IL-23 or IFN-γ. Typically, theantagonist is an antibody. Typically, the antibody is a monoclonalantibody.

In a sixth aspect, the present invention provides a compositioncomprising CD4⁺,CD25⁺ T cells activated to a specific antigen togetherwith a cytokine and/or pharmaceutically acceptable carrier wherein saidCD4⁺,CD25⁺ T cells activated to a specific antigen have been cultured inthe presence of at least one cytokine selected from the group consistingof IL-5, IL-12, IL-23 and IFN-γ, or a biologically active fragmentthereof, or a functionally equivalent molecule thereof.

In a seventh aspect, the invention provides a method for treating orpreventing in a subject in need thereof a disease resulting from animmune response to a specific antigen, the method comprising the step ofadministering to the subject a therapeutically effective amount ofCD4⁺,CD25⁺ T cells activated to the specific antigen grown in vitro byculturing CD4⁺,CD25⁺ T cells activated to the specific antigen in vitroin the presence of at least one cytokine selected from the groupconsisting of IL-5, IL-12, IL-23 and IFN-γ, or a biologically activefragment thereof, or a functionally equivalent molecule thereof.

In one embodiment, the number of CD4⁺ T cells in the subject is reducedprior to administering the CD4⁺,CD25⁺ T cells activated to the specificantigen. Typically, the number of CD4⁺,CD25⁻ T cells in the subject isreduced. The number of CD4⁺ T cells may be reduced by any methods knownin the art for reducing lymphocytes in a subject including irradiation,administration of antibodies against CD4, typically CD4⁺,CD25⁻ T cells,such as anti-CD3, anti-CD4, anti-CD45RB/RO, or administration ofcytotoxic or alkylating agent therapy.

In one embodiment, the method comprises the further step of contactingthe CD4⁺,CD25⁺ T cells activated to the specific antigen with thespecific antigen in vitro. The CD4⁺,CD25⁺ T cells activated to thespecific antigen may be contacted with the specific antigen in vitroprior to, or simultaneous with, culturing the T cells in the presence ofthe at least one cytokine selected from the group consisting of IL-5,IL-12, IL-23 and IFN-γ, or a biologically active fragment thereof, or afunctionally equivalent molecule thereof. Typically, the T cells arecultured in the presence of the at least one cytokine selected from thegroup consisting of IL-5, IL-12, IL-23 and IFN-γ, or a biologicallyactive fragment thereof, or a functionally equivalent molecule thereof,after contacting the lymphocyte with the specific antigen.

The disease may be any disease resulting from an immune response to oneor more specific antigens. In one embodiment, the disease is associatedwith an immune response to an autoantigen, for example an autoimmunedisease. Examples of the types of autoimmune disease that may beprevented or treated using the method of the present invention include,for example, type 1 insulin dependent diabetes mellitis, inflammatorybowel syndrome including ulcerative colitis and Crohn's disease,thrombotic thrombocytopenic purpura, Sjogren's syndrome, encephalitis,acute disseminated encephalomyelitis, Guillain Barre Syndrome, chronicinflammatory demyelination polyneuropathy, idiopathic pulmonaryfibrosis/alveolitis, asthma, uveitis, iritis, optic neuritis, rheumaticfever, Reiter's syndrome, psoriasis, psoriatic arthritis, multiplesclerosis, progressive systemic sclerosis, primary biliary cirrhosis,pemphigus, pemphigoid, necrotising vasculitis, myasthenia gravis,polymyositis, sarcoidosis, granulomatosis, vasculitis, perniciousanemia, central nervous system inflammatory disorder, autoimmunehaemolytic anaemia, Hashimoto's thyroiditis, Graves disease, habitualspontaneous abortions, Raynaud's syndrome, dermatomyositis; chronicactive hepatitis, celiac disease, autoimmune complications of AIDS,atrophic gastritis, ankylosing spondylitis, Addison's disease, chronicdemyelinating neuropathy, glomerulonephritis including membranousnephropathy, focal sclerosing glomerulonephritis and minimal changenephropathy, systemic lupus erythematosis, scleroderma, rheumatoidarthritis, juvenile arthritis.

In another embodiment, the disease is the result of an immune responseto a non-self antigen in contact with the subject. This may be the casefollowing, for example, transplantation of cells or tissue to thesubject where the transplanted cells or tissue undergoes rejection bythe immune system of the subject. Typically, the transplanted cells ortissue is allograft or xenograft cells or tissue. Examples of transplantcells or tissue include kidney, liver, heart, valves, lung, skin,pancreas, cornea, lens, bone marrow, muscle, connective tissue, vasculartissue, gastrointestinal tissue, nervous tissue, bone, stem cells orgenetically modified cells.

Genetically modified cells may be, for example, stem cells, that havebeen transfected with a therapeutic or other agent.

Alternatively, the disease may be the result of an immune response to anallergen in contact with the subject. Examples of diseases resultingfrom an allergen include asthma, eczema, atopic dermatitis, anaphylaxis,hayfever, allergic conjunctivitis, contact dermatitis, food allergy.

In an eighth aspect, the invention provides a composition when used forreducing or eliminating tolerance to a specific antigen in a subject,the composition comprising an antagonist of one or more of the cytokinesselected from the group consisting of IL-5, IL-12, IL-23 and IFN-γ, or abiologically active fragment thereof, or a functionally equivalentmolecule thereof.

The antagonist may be any molecule which reduces or eliminates theactivity of IL-5, IL-12, IL-23 and/or IFN-γ, biologically activefragments thereof, or functionally equivalent molecules thereof.Examples of suitable antagonists include antibody receptor molecules,antisense molecules, iRNA or siRNA molecules, or any other moleculeswhich reduce or eliminate the biological activity of IL-5, IL-12, IL-23or IFN-γ, biologically active fragments thereof, or functionallyequivalent molecules thereof. Typically, the antibody is a monoclonalantibody.

In a ninth aspect, the invention provides a method for treating orpreventing in a subject in need thereof a disease resulting fromtolerance to a specific antigen, the method comprising the step ofadministering to the subject a therapeutically effective amount of anantagonist of one or more cytokines selected from the group consistingof IL-5, IL-12, IL-23 and IFN-7, or a biologically active fragmentthereof, or a functionally equivalent molecule thereof.

In one embodiment, the method may further comprise the step ofadministering an effective amount of an antibody against CD4⁺,CD25⁺ Tcells. Typically, the antibody is an anti-CD25 antibody.

The disease may be any disease resulting from tolerance to a specificantigen. Examples of such disease include cancer, chronic infection suchas hepatitis B and C, leprosy, tuberculosis, cryptococcosis, herpessimplex.

The invention also contemplates a kit for use with the methods of theinvention. A kit for assessing whether a subject comprises CD4⁺,CD25⁺ Tcells capable of imparting tolerance to a specific antigen may comprisemedia and/or cytokines for incubating the sample of lymphocytes. Thecytokines may be one or more of IL-5, IL-12, IL-23 or IFN-γ, or abiologically active fragment thereof, or a functionally equivalentmolecule thereof.

A kit for increasing tolerance to a specific antigen may comprise one ormore cytokines selected from the group consisting of IL-5, IL-12, IL-23and IFN-γ, or a biologically active fragment thereof, or a functionallyequivalent molecule thereof. The kit may further comprise a specificantigen in a form suitable for contacting CD4⁺,CD25⁺ T cells from asubject in vitro. For example, the kit may comprise a specific antigenon the surface of an antigen presenting cell. It will be appreciated bypersons skilled in the art that the relevant part of the specificantigen may be incorporated into an appropriate MHC molecule on thesurface of the antigen presenting cell. The kit may further compriseIL-13, a biologically active fragment thereof, or a functionallyequivalent molecule thereof.

A kit for decreasing tolerance to a specific antigen may comprise anantagonist of one or more cytokines selected from the group consistingof IL-5, IL-12, IL-23 and IFN-γ, or a biologically active fragmentthereof, or a functionally equivalent molecule thereof. The antagonistmay be any molecule which reduces or eliminates the activity of IL-5,IL-12, IL-23 and/or IFN-γ, biologically active fragments thereof, orfunctionally equivalent molecules thereof. Examples of suitableantagonists include antibody receptor molecules, antisense molecules,iRNA or siRNA molecules, or any other molecules which reduce oreliminate the biological activity of IL-5, IL-12, IL-23 or IFN-γ,biologically active fragments thereof, or functionally equivalentmolecules thereof. Typically, the antibodies are monoclonal antibodies.The kit may further comprise IL-2, a biologically active fragmentthereof, or a functionally equivalent molecule thereof, and/or IL-4, abiologically active fragment thereof, or a functionally equivalentmolecule thereof.

The kits described above may further comprise instructions for use ofthe kit.

The subject may be any subject which produces CD4⁺,CD25⁺ T cells. Thesubject may be a mammal. The mammal may be a human or non-human animal,such as rodent, non-human primate, cattle, pig, sheep, camel, goat, cat,dog or horse. Typically, the subject is a human.

In a tenth aspect, the invention provides a method of inducing toleranceto a specific antigen in a subject in need thereof, wherein thesubject's immune system is exposed to the specific antigen, the methodcomprising administering to the subject:

-   -   (i) an effective amount of one or more cytokines selected from        the group consisting of IL-2, a biologically active fragment        thereof, or a functionally equivalent molecule thereof, and        IL-4, a biologically active fragment thereof, or a functionally        equivalent molecule thereof; and    -   (ii) an effective amount of at least one cytokine selected from        the group consisting of IL-5, IL-12, IL-23 and IFN-γ, or a        biologically active fragment thereof, or a functionally        equivalent molecule thereof.

It is envisaged that administration of IL-2 and/or IL-4, or abiologically active fragment thereof, or a functionally equivalentmolecule thereof, supports activation of naïve CD4⁺,CD25⁺ T cells incontact with the specific antigen, and the at least one cytokineselected from the group consisting of IL-5, IL-12, IL-23 and IFN-γ, or abiologically active fragment thereof, or a functionally equivalentmolecule thereof, maintains and/or stimulates proliferation ofCD4⁺,CD25⁺ T cells activated to a specific antigen.

The at least one cytokine of step (ii) may be administeredsimultaneously with, or subsequent to, administration of the one or morecytokines of step (i). Suitably, the at least one cytokine of step (ii)is administered subsequent to administration of the one or morecytokines of step (i). Typically, the at least one cytokine of step (ii)is administered between 24 hours and 1 week following administration ofthe one or more cytokines of step (i).

In one embodiment of the tenth aspect, the method comprisesadministering an effective amount of IL-2, a biologically activefragment thereof, or a functionally equivalent molecule thereof, and aneffective amount of IL-12, IL-23 and/or IFN-γ, a biologically activefragment thereof, or a functionally equivalent molecule thereof.

In another embodiment of the tenth aspect, the method comprisesadministering an effective amount of IL-4, a biologically activefragment thereof, or a functionally equivalent molecule thereof, and aneffective amount of IL-5, a biologically active fragment thereof, or afunctionally equivalent molecule thereof.

In yet another embodiment of the tenth aspect, the method comprisesadministering an effective amount of IL-2, a biologically activefragment thereof, or a functionally equivalent molecule thereof, and/orIL-4, a biologically active fragment thereof, or a functionallyequivalent molecule thereof, and an effective amount of at least onecytokine selected from the group consisting of IL-5, IL-12, IL-23 andIFN-γ, or a biologically active fragment thereof, or a functionallyequivalent molecule thereof.

In another embodiment of the tenth aspect, the method further comprisesthe step of administering an effective amount of IL-13, a biologicallyactive fragment thereof, or a functionally equivalent molecule thereof.

In an eleventh aspect, the invention provides a method of treating orpreventing in a subject in need thereof a disease resulting from animmune response to an antigen, the method comprising administering tothe subject:

-   -   (i) an effective amount of one or more cytokines selected from        the group consisting of IL-2, a biologically active fragment        thereof, or a functionally equivalent molecule thereof, and        IL-4, a biologically active fragment thereof, or a functionally        equivalent molecule thereof; and    -   (ii) an effective amount of at least one cytokine selected from        the group consisting of IL-5, IL-12, IL-23 and IFN-γ, or a        biologically active fragment thereof, or a functionally        equivalent molecule thereof.

The at least one cytokine of step (ii) may be administeredsimultaneously with, or subsequent to, administration of the one or morecytokines of step (i). Suitably, the at least one cytokine of step (ii)is administered subsequent to administration of one or more cytokines ofstep (i). Typically, the at least one cytokine of step (ii) isadministered between 24 hours and 1 week following administration of theone or more cytokines of step (i).

In one embodiment of the eleventh aspect, the method comprisesadministering an effective amount of IL-2, or a biologically activefragment thereof, or a functionally equivalent molecule thereof, and aneffective amount of at least one cytokine selected from the groupconsisting of IL-12, IL-23 and IFN-γ, or a biologically active fragmentthereof, or a functionally equivalent molecule thereof.

In another embodiment of the eleventh aspect, the method comprisesadministering an effective amount of IL-4, a biologically activefragment thereof, or a functionally equivalent molecule thereof, and aneffective amount of IL-5, a biologically active fragment thereof, or afunctionally equivalent molecule thereof.

In yet another embodiment of the eleventh aspect, the method comprisesadministering an effective amount of IL-2, a biologically activefragment thereof, or a functionally equivalent molecule thereof, and/orIL-4, a biologically active fragment thereof, or a functionallyequivalent molecule thereof, and at least one cytokine selected from thegroup consisting of IL-5, IL-12, IL-23 and IFN-γ, or a biologicallyactive fragment thereof, or a functionally equivalent molecule thereof.

In another embodiment of the eleventh aspect, the method furthercomprises the step of administering an effective amount of IL-13, abiologically active fragment thereof, or a functionally equivalentmolecule thereof.

In one embodiment of the eleventh aspect, the method may comprise, priorto step (ii), the steps of:

-   (a) obtaining from the subject a sample of lymphocytes comprising    CD4⁺,CD25⁺ T cells; and-   (b) determining whether the sample comprises CD4⁺,CD25⁺ T cells that    are responsive to one or more cytokines selected from the group    consisting of IL-5, IL-12, IL-23 and IFN-γ, or a biologically active    fragment thereof, or a functionally equivalent molecule thereof.

In such embodiments, step (ii) typically comprises administering aneffective amount of a cytokine selected from the group consisting ofIL-5, IL-12, IL-23 and IFN-γ, or a biologically active fragment thereof,or a functionally equivalent molecule thereof, to which the CD4⁺,CD25⁺ Tcells have been determined to be responsive.

In one embodiment, responsiveness of the CD4⁺,CD25⁺ T cells from thesample to the one or more cytokines is determined by detecting theexpression of a receptor for the one or more cytokines. Expression ofthe receptor for the one or more cytokines may be detected using anantibody to the receptor. Expression of the receptor for the one or morecytokines may be detected by detecting expression of RNA for thereceptor. For example, assay using PCR, RT-PCR, northern blot analysisetc. may be used to detect mRNA that encodes the receptor for the one ormore cytokines.

In another embodiment, responsiveness to the one or more cytokines maybe determined by assessing proliferation of the sample in the presenceof the one or more cytokines selected from the group consisting of IL-5,IL-12, IL-23 and IFN-γ, or a biologically active fragment thereof, or afunctionally equivalent molecule thereof. For example, the sample may beincubated in the presence of the one or more cytokines selected from thegroup consisting of IL-5, IL-12, IL-23 and IFN-γ, or a biologicallyactive fragment thereof, or a functionally equivalent molecule thereof,typically also in the presence of the specific antigen, wherebyproliferation of CD4⁺,CD25⁺ T cells in the presence of the one or morecytokines indicates that the CD4⁺,CD25⁺ T cells are responsive to theone or more cytokines selected from the group consisting of IL-5, IL-12,IL-23 and IFN-γ, or a biologically active fragment thereof, or afunctionally equivalent molecule thereof.

The disease may be any disease resulting from an immune response to oneor more specific antigens. In one embodiment, the disease is associatedwith an immune response to an autoantigen, for example an autoimmunedisease. Examples of the types of autoimmune disease that may beprevented or treated using the method of the present invention include,for example, type 1 insulin dependent diabetes mellitis, inflammatorybowel syndrome including ulcerative colitis and Crohn's disease,thrombotic thrombocytopenic purpura, Sjogren's syndrome, encephalitis,acute disseminated encephalomyelitis, Guillain Barre Syndrome, chronicinflammatory demyelination polyneuropathy, idiopathic pulmonaryfibrosis/alveolitis, asthma, uveitis, iritis, optic neuritis, rheumaticfever, Reiter's syndrome, psoriasis, psoriatic arthritis, multiplesclerosis, progressive systemic sclerosis, primary biliary cirrhosis,pemphigus, pemphigoid, necrotising vasculitis, myasthenia gravis,polymyositis, sarcoidosis, granulomatosis, vasculitis, perniciousanemia, central nervous system inflammatory disorder, autoimmunehaemolytic anaemia, Hashimoto's thyroiditis, Graves disease, habitualspontaneous abortions, Raynaud's syndrome, dermatomyositis, chronicactive hepatitis, celiac disease, autoimmune complications of AIDS,atrophic gastritis, ankylosing spondylitis, Addison's disease, chronicdemyelinating neuropathy, glomerulonephritis including membranousnephropathy, focal sclerosing glomerulonephritis and minimal changenephropathy, systemic lupus erythematosis, scleroderma, rheumatoidarthritis, juvenile arthritis.

In another embodiment, the disease is the result of an immune responseto a non-self antigen in contact with the subject. This may be the casefollowing, for example, transplantation of cells or tissue to thesubject where the transplanted cells or tissue undergoes rejection bythe immune system of the subject. Typically, the transplanted cells ortissue is allograft or xenograft cells or tissue. Examples of transplantcells or tissue include kidney, liver, heart, valves, lung, skin,pancreas, cornea, lens, bone marrow, muscle, connective tissue, vasculartissue, gastrointestinal tissue, nervous tissue, bone, stem cells orgenetically modified cells. Genetically modified cells may be, forexample, autologous cells, including skin cells, which have beentransfected with a therapeutic or other agent.

Alternatively, the disease may be the result of an immune response to anallergen in contact with the subject. Examples of diseases resultingfrom an allergen include asthma, eczema, atopic dermatitis, anaphylaxis,hayfever, allergic conjunctivitis, contact dermatitis, food allergy,drug or other chemical allergy, venom allergy.

In a twelfth aspect, the invention provides a method of inducingtolerance to a specific antigen in a subject in need thereof, comprisingadministering to the subject an effective amount of one or morecytokines selected from the group consisting of IL-5, IL-12, IL-23 andIFN-γ, or a biologically active fragment thereof, or a functionallyequivalent molecule thereof.

Typically, the method does not comprise administering anti CD-3antibody.

In some embodiments the subject's immune system is exposed to thespecific antigen. In embodiments where the subject's immune system isexposed to the specific antigen, the method may comprise:

-   -   (a) obtaining from the subject a sample of lymphocytes        comprising CD4⁺,CD25⁺ T cells;    -   (b) determining whether the sample comprises CD4⁺,CD25⁺ T cells        that are responsive to one or more cytokines selected from the        group consisting of IL-5, IL-12, IL-23 and IFN-γ, or a        biologically active fragment thereof, or a functionally        equivalent molecule thereof; and    -   (c) administering an effective amount of the one or more        cytokines selected from the group consisting of IL-5, IL-12,        IL-23 and IFN-γ, or a biologically active fragment thereof, or a        functionally equivalent molecule thereof, to which the        CD4⁺,CD25⁺ T cells are responsive.

In a thirteenth aspect, the invention provides a method of treating orpreventing in a subject in need thereof a disease resulting from animmune response to a specific antigen, the method comprisingadministering an effective amount of one or more cytokines selected fromthe group consisting of IL-5, IL-12, IL-23 and IFN-γ, or a biologicallyactive fragment thereof, or a functionally equivalent molecule thereof.

In one embodiment, the method comprises the steps of:

-   -   (a) obtaining from the subject a sample of lymphocytes        comprising CD4⁺,CD25⁺ T cells;    -   (b) determining whether the sample comprises CD4⁺,CD25⁺ T cells        that are responsive to one or more cytokines selected from the        group consisting of IL-5, IL-12, IL-23 and IFN-γ, or a        biologically active fragment thereof, or a functionally        equivalent molecule thereof; and    -   (c) administering an effective amount of the one or more        cytokines selected from the group consisting of IL-5, IL-12,        IL-23 and IFN-γ, or a biologically active fragment thereof, or a        functionally equivalent molecule thereof, to which the        CD4⁺,CD25⁺ T cells are responsive.

The responsiveness of the CD4⁺,CD25⁺ T cells from the sample to the oneor more cytokines may be determined by detecting the expression of areceptor for the one or more cytokines. Expression of the receptor forthe one or more cytokines may be detected using an antibody to thereceptor. Expression of the receptor for the one or more cytokinesselected from the group consisting of IL-5, IL-12, IL-23 and IFN-γ, or abiologically active fragment thereof, or a functionally equivalentmolecule thereof may be detected by detecting expression of RNA for thereceptor. For example, assay using PCR, RT-PCR, northern blot analysisetc. may be used to detect mRNA that encodes the receptor for the one ormore cytokines selected from the group consisting of IL-5, IL-12 andIFN-γ, or a biologically active fragment thereof, or a functionallyequivalent molecule thereof.

The responsiveness to the one or more cytokines selected from the groupconsisting of IL-5, IL-12, IL-23 and IFN-γ, or a biologically activefragment thereof, or a functionally equivalent molecule thereof may bedetermined by assessing proliferation of the sample in the presence ofthe one or more cytokines selected from the group consisting of IL-5,IL-12, IL-23 and IFN-γ, or a biologically active fragment thereof, or afunctionally equivalent molecule thereof. For example, the sample may beincubated in the presence of the one or more cytokines selected from thegroup consisting of IL-5, IL-12, IL-23 and IFN-γ, or a biologicallyactive fragment thereof, or a functionally equivalent molecule thereof,typically also in the presence of the specific antigen, wherebyproliferation of CD4⁺,CD25⁺ T cells in the presence of the one or morecytokines selected from the group consisting of IL-5, IL-12, IL-23 andIFN-γ, or a biologically active fragment thereof, or a functionallyequivalent molecule thereof indicates that the CD4⁺,CD25⁺ T cells areresponsive to the one or more cytokines selected from the groupconsisting of IL-5, IL-12, IL-23 and IFN-γ, or a biologically activefragment thereof, or a functionally equivalent molecule thereof.

In one embodiment, the method comprises the step, prior to orsimultaneously with administering the one or more cytokines selectedfrom the group consisting of IL-5, IL-12, IL-23 and IFN-γ, or abiologically active fragments thereof, or a functionally equivalentmolecule thereof, the step of administering to the subject an effectiveamount of one or more cytokines selected from the group consisting ofIL-2 and IL-4, biologically active fragments thereof, and functionallyequivalent molecules thereof.

In one embodiment of the twelfth or thirteenth aspect, the methodfurther comprises the step of administering an effective amount ofIL-13, a biologically active fragment thereof, or a functionallyequivalent molecule thereof.

The disease resulting from an immune response to an antigen may be anyof the diseases resulting from an immune response listed above.

In a fourteenth aspect, the invention provides a kit when used with themethod of the ninth, tenth or twelfth aspect, the kit comprising IL-2, abiologically active fragment thereof, or a functionally equivalentmolecule thereof, and/or IL-4, a biologically active fragment thereof,or a functionally equivalent molecule thereof, and/or at least onecytokine selected from the group consisting of IL-5, IL-12, IL-23 andIFN-γ, or a biologically active fragment thereof, or a functionallyequivalent molecule thereof. The kit may further comprise IL-13. The kitmay further comprise instructions for use.

It is also envisaged that any of the above methods for increasingtolerance to a specific antigen may be applied in methods of treatmentof subjects which involve introducing a foreign entity into the subjectfor delivery of an agent, typically a therapeutic agent. For example,the methods of the invention may be used to increase tolerance to avirus used for gene therapy, or to other gene delivery vehicles whichotherwise elicit an immune response.

In a fifteen aspect, the invention provides the use of CD4⁺,CD25⁺ Tcells activated to a specific antigen grown in vitro by the method ofthe third aspect in the manufacture of a medicament for increasingtolerance in a subject in need thereof.

In a sixteenth aspect, the invention provides the use of an antagonistof IL-5, IL-12, IL-23 or IFN-7 in the manufacture of a medicament forreducing tolerance to a specific antigen in a subject.

In a seventeenth aspect, the invention provides the use of CD4⁺,CD25⁺ Tcells activated to a specific antigen grown in vitro by the method ofthe third aspect in the manufacture of a medicament for treating orpreventing in a subject in need thereof a disease resulting from animmune response to the specific antigen.

In an eighteenth aspect, the invention provides the use of CD4⁺,CD25⁺ Tcells activated to a specific antigen grown in vitro by the method ofthe third aspect in the manufacture of a medicament for treating orpreventing in a subject in need thereof a disease resulting fromtolerance to the specific antigen.

In a nineteenth aspect, the invention provides the use of one or morecytokines selected from the group consisting of IL2, IL-4, abiologically active fragment thereof, or a functionally equivalentmolecule thereof, in the manufacture of a medicament for the treatmentof a subject to induce tolerance in the subject, the treatmentcomprising administering an effective amount of the medicament and aneffective amount of at least one cytokine selected from the groupconsisting of IL-5, IL-12, IL-23 and IFN-γ, a biologically activefragment thereof, or a functionally equivalent molecule thereof.

In a twentieth aspect, the invention provides the use of one or morecytokines selected from the group consisting of IL-5, IL-12, IL-23 andIFN-γ, a biologically active fragment thereof, or a functionallyequivalent molecule thereof, in the manufacture of a medicament for thetreatment of a subject to induce tolerance in the subject, the treatmentcomprising administering an effective amount of the medicament and aneffective amount of at least one cytokine selected from the groupconsisting of IL-2 and IL-4, a biologically active fragment thereof, ora functionally equivalent molecule thereof.

In a twenty-first aspect, the invention provides the use of one or morecytokines selected from the group consisting of IL-5, IL-12, IL-23 andIFN-γ, a biologically active fragment thereof, or a functionallyequivalent molecule thereof, in the manufacture of a medicament forincreasing the tolerance to a subject in need thereof.

In a twenty-second aspect, the invention provides the use of one or morecytokines selected from the group consisting of IL-5, IL-12, IL-23 andIFN-γ, a biologically active fragment thereof, or a functionallyequivalent molecule thereof, in the manufacture of a medicament fortreating or preventing in a subject in need thereof a disease resultingfrom an immune response to a specific antigen.

In a twenty-third aspect, the invention provides a method of activatingCD4⁺,CD25⁺ T cells to a specific antigen, the method comprising:

-   -   (a) contacting naïve CD4⁺,CD25⁺ T cells with the specific        antigen; and    -   (b) culturing the CD4⁺,CD25⁺ T cells in the presence of IL-2, a        biologically active fragment thereof, or a functionally        equivalent molecule thereof, and/or IL-4, a biologically active        fragment thereof, or a functionally equivalent molecule thereof.

In a twenty-fourth aspect, the invention provide a method of culturingCD4⁺,CD25⁺ T cells activated to a specific antigen; the methodcomprising culturing the CD4⁺,CD25⁺ T cells in the presence of at leastone cytokine selected from the group consisting of IL-5, IL-12, IL-23and IFN-γ, a biologically active fragment thereof, or a functionallyequivalent molecule thereof.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A is a graph of the proliferation of naïve unfractionatedlymphocytes (dashed line and filled in circles), naïve CD4⁺ Tcell/lymphocytes (hard line, filled in circles), naïve CD4⁺,CD25⁺ Tcells (open circles thin line), and naïve CD4⁺,CD25⁻ T cells (opencircles thick line). Proliferation assayed at days 2, 3, 4, 5 and 6following contacting the lymphocytes with an alloantigen. FIG. 1B is agraph comparing proliferation of naïve CD4⁺ T cell lymphocytes, naveCD4⁺,CD25⁺ T cells and naïve CD4⁺,CD25⁻ T cells in response to selfantigen (black), PVG antigen (donor antigen)(light grey) and Lewisantigen (third party antigen) (dark grey) after 4 days.

FIG. 1C is a graph of the proliferation (on y axis) of serial dilutions(shown on x axis) of populations of naïve CD4⁺ T cell lymphocytes(triangles), naïve CD4⁺,CD25⁺ T cells (squares) and naïve CD4⁺,CD25⁻ Tcells (circles) following contacting the lymphocytes with alloantigen(PVG).

FIG. 1D is a graph of the effect on proliferation of mixing separatenave CD4⁺,CD25⁺ T cells with separate CD4⁺,CD25⁻ T cells over 6 daysfollowing contacting the lymphocytes with alloantigen (PVG). Mixturesare as indicated.

FIG. 1E is graphs of proliferation of CD4⁺ T cells, CD4⁺,CD25⁺ T cellsand CD4⁺,CD25⁻ T cells from DA rats tolerant to a PVG cardiac allograftfollowing contacting the lymphocytes with self antigen (black), donorantigen (PVG antigen—light grey) or third party antigen (Lewis—darkgrey).

FIG. 1F is graphs showing the results of a limiting dilution assay ofunfractionated T cells, fractionated CD4⁺ T cells and fractionatedCD4⁺,CD25⁻ T cells from DA rats tolerant to a PVG allograft at day 4following contacting the lymphocytes with self antigen (open circles,full thin line), donor antigen (dashed line) or third party antigen(thick line, open squares).

FIG. 2A is a graph of proliferation of naïve CD4⁺,CD25⁺ T cellsfollowing contact with self antigen (black) or donor antigen(cross-hatch) and incubation in the presence of cytokines as indicated.

FIG. 2B is a graph of proliferation at day 3 of activated CD4⁺,CD25⁺ Tcells against self antigen (black) or donor antigen (grey) in thepresence of TGF-β(D), IFN-γ(E), IL-12(p7O) (F), IL-5(G) or IL-10(H).

FIG. 3A is a graph of rejection time of heart allografts in ratsfollowing whole-body irradiation and administration of various doses (asindicated at A to D) of naïve CD4⁺ T cells/lymphocytes.

FIG. 3B is a graph of heart graft function up to 50 dayspost-transplantation in rats following administration of A, 5×10⁶ naïveCD4⁺ T cell lymphocytes (closed triangles); B, 20×10⁶ naïve CD4⁺ T cells(closed circles); C, 5×10⁶ naïve CD4⁺,CD25⁻ T cells (open triangles); D,0.5×10⁶ naïve CD4⁺,CD25⁺ T cells plus 5×10⁶ naïve CD4⁺ T cells (opensquares); E, 5×10⁶ naïve dDe,CD25⁺ T cells plus 5×10⁶ naïve CD4⁺ T cells(closed squares).

FIG. 4A is graphs showing the effect of IL-2 (upper) and IL-4 (lower) onproliferation of naïve CD4⁺,CD25⁺ T cells from DA rats alone (black), incontact with self antigen (cross-hatch) or in contact with alloantigen(PVG) (shaded) following 4 and 5 days after contact with antigen asindicated.

FIG. 4B is a graph showing the effect from day 3 to day 6 onproliferation of naïve CD4⁺,CD25⁺ T cells cultured with alloantigen andno cytokines (dark shading), IL-2 (mid-shading) or IL-4 (light shading).

FIG. 4C shows the results of semi-quantitative RT-PCR using primers tocytokines or cytokine receptors (as indicated) on mRNA isolated fromnaïve CD4⁺,CD25⁺ T cells cultured in the presence of self or alloantigenand IL-2 or IL-4 as indicated.

FIG. 4D shows the results of semi-quantitative RT-PCR using primers tocytokines or cytokine receptors (as indicated) on mRNA isolated fromCD4⁺,CD25⁺ T cells from DA rats tolerant to PVG allografts cultured inthe presence of self antigen (from DA rats), donor antigen (from PVGrats) or third party antigen (from Lewis rats) in the presence of IL-2or IL-4.

FIG. 4E shows the results of semi-quantitative RT-PCR using primers tothe IL-5 receptor alpha chain or to GADPH on mRNA isolated from naïveCD4⁺,CD25⁺ T cells incubated in the presence of either self stimulatorsor PVG stimulators, alone or with either IL-2 or IL-4.

FIG. 4F is a graph showing the results of real-time RT-PCR using primersto the IFN-γ receptor or to GADPH on mRNA isolated from nave CD4⁺,CD25⁺T cells incubated in the presence of self or alloantigen, and with nocytokine or with either IL-2 or IL-4.

FIG. 5A shows the results of semi-quantitative RT-PCR using primers tocytokine mRNA isolated from CD4⁺,CD25⁺ T cells and CD4⁺CD25⁻ T cellsalone or admixed in a 1:1 ratio. All populations were from naïve DA ratsand were incubated in the presence of self or alloantigen.

FIG. 5B is graphs showing proliferation of CD4⁺,CD25⁺ T cells,CD4⁺,CD25⁻ T cells and CD4⁺,CD25⁻ T cells admixed with CD4⁺,CD25⁺ Tcells in a ratio of 1:1 following incubation in the presence of selfantigen (black) or alloantigen (grey) The effects of antibodies to blockIL-5, TGF-β or IL-10 were compared to a control antibody Mog-Ig2a.

FIG. 6A is graphs showing the survival of heterotopic cardiac allograftstransplanted from PVG rats or Lewis rats into irradiated DA rats thatwere adoptively restore with CD4⁺ T cells from DA rats tolerant to a PVGheart allografts where the cells had been cultured for 3 days with PVGstimulator cells in media supplemented with either IL-4 (dotted line) orIL-5 (solid line).

FIG. 6B are graphs showing the proliferation of CD4⁺,CD25⁺ T cells andCD4⁺,CD25⁻ T cells from DA rats tolerant to PVG allografts followingincubating the lymphocytes with PVG antigen in the presence or absenceof IL-4 (as indicated), followed by incubating the cells alone (white),or in the presence of self antigen (DA) (dark grey), donor antigen(PVG)(black) or third party antigen (Lewis) (light grey), and in thepresence or absence of IL-4.

FIG. 7 is a graph showing the proliferation of unfractionated navelymphocytes from DA rats tolerant to PVG allografts at day 4 followingcontact with self-antigen or alloantigen in the presence of cytokines asindicated.

FIG. 8A is a graph showing the clinical time course of ExperimentalAllergic Neuritis (EAN) in Lewis rats following administration oflymphocytes from tolerant (recovered) Lewis rats. Closed squares areuntreated rats, closed circles are rats treated with CD4⁴⁺,CD25⁺ T cellsfrom rats tolerant to EAN and open circles are rats treated withCD4⁺,CD25⁻ T cells from rats tolerant to EAN.

FIG. 8B is a graph showing the weight change over a time course ofExperimental Allergic Neuritis (EAN) in Lewis rats followingadministration of lymphocytes from tolerant (recovered) Lewis rats.Closed squares are untreated rats, closed circles are rats treated withCD4⁺,CD25⁺ T cells and open circles are rats treated with CD4⁺,CD25⁻ Tcells.

FIG. 9A is a graph showing the proliferation of various combinations oflymphocytes (as indicated) from naïve Lewis rats in response to PNM.

FIG. 9B is a graph showing the proliferation of various combinations oflymphocytes (as indicated) from Lewis rats tolerant to PNM antigen(recovered from the EAN 4 weeks after immunisation) in response to PNMantigen.

FIG. 10 shows the % survival in PVG cardiac allografts (upper panel) andLewis (third party) cardiac allografts (lower panel) over 50 days inirradiated DA rats which received 20×10⁶ CD4⁺ T cells from DA ratstolerant to PVG antigen cultured with IFN-γ in the presence of PVGantigen (full line, upper graph) (dotted line, lower graph); 5×10⁶ NaïveCD4⁺ T cells (thick dashed line, upper and lower graph); 20×10⁶ NaïveCD4⁺ T cells (thin dashed line, upper graph); 20×10⁶ CD4⁺,CD25⁺ T cellsfrom DA rats tolerant to PVG antigen mixed with 5×10⁶ Naïve CD4⁺ T cells(dotted line, upper graph); irradiated DA rats (no cells) (solid line,upper and lower graph).

FIG. 11 shows the effect on proliferation of CD4⁺,CD25⁺ T cells ofincubating CD4⁺ T cells (top row), CD4⁺,CD25⁻ T cells (middle row) orCD4⁺,CD25⁺ T cells (bottom row) in the presence of autoantigen from DArats (left column of graphs) or alloantigen (from PVG rats) (rightcolumn of graphs) in the presence of IL-2, IL-12, or IL-2 and IL-12 asindicated at the bottom of the diagram.

FIG. 12 shows proliferation of CD4⁺,CD25⁺ T cells after activation inthe presence of IL-2 and PVG antigen for 3 days, followed by incubationin the presence of no supplement (Resp. Stimul), CHO-K supernatant(CHO-K), IL-2 (IL-2), IL-12p70 (IL-12p70) or IL-12p40 (IL-12p40).

FIG. 13 shows the results of RT-PCR GAPDH, of IL-2 or IL-12Rβ2 mRNA fromCD4⁺,CD25⁺ T cells following culturing with autoantigen (DA antigen) oralloantigen (PVG antigen) for 4 days in the presence of IL-2 or IL-4.

FIG. 14 shows the effect of administration of IL-5 on severity (A) andweight loss (B) in an experiment disease model of EAN. Open squares=Ratsadministered IL-5; closed circles=rats administered CHO-K supernatant;open circles=no treatment.

FIG. 15 shows the percent of demyelinated nerve fibres in peripheralnerves from an experimental rat model of BAN 14 and 21 days afterimmunisation with PNM. Rats treated with IL-5 at immunisation arerepresented by grey bars, rats that were untreated are represented byblack bars.

FIG. 16 shows the normalised copy number of mRNA following real timeRT-PCR from draining lymph nodes (LN) or cauda equina (CE) of rats at 14days or 21 days post-immunisation with PNM using primers to TCR, IFN-γ,IL-2, IL-10 or TNF-α (as indicated). Black bars represent IL-5 treated,open bars represent untreated. Significant is indicated as * p<0.05 and** p<0.01.

FIG. 17 shows the results of RT-PCR analysis of IL-5, IL-5Rα, IL-4 andIL-13 mRNA expression at day 14 and day 21 in draining lymph nodes (LN)and cauda equine (CE) of rats immunised with DNA and either administeredIL-5 (IL-5) or no IL-5 (CTL).

FIG. 18 shows the effect of CD4⁺,CD25⁺ T cells on proliferation of naïveCD4⁺,CD25⁻ T cells in a limiting dilution assay, A: naïve CD4⁺,CD25⁻ Tcells were mixed with 1:2 serial dilution of naïve CD4⁺ CD25⁻ T cells;B: naïve CD4⁺,CD25⁻ T cells were mixed with 1:2 serial dilution ofCD4⁺,CD25⁺ T cells which had been cultured in the presence of PVGantigen and IL-2.

FIG. 19 shows the effect of CD4⁺,CD25⁺ T cells on proliferation of naïveCD4⁺,CD25⁻ T cells in a limiting dilution assay, A: naïve CD4⁺,CD25⁻ Tcells were mixed with 1:2 serial dilution of naïve CD4⁺,CD25⁺ T cells;B: naïve CD4⁺,CD25⁻ T cells were mixed with 1:2 serial dilution ofCD4⁺,CD25⁺ T cells which had been cultured in the presence of PVGantigen and IL-4.

FIG. 20 shows the expected relative cell proliferation of isolatedCD4⁺,CD25⁺ T cells from a subject tolerant (A and C) or non-tolerant (Band D) to a donor antigen following culturing in the absence (A and B)or presence (C and D) of IL-5, IL-12 or IFN-γ and self antigen (S),donor antigen (D) or third party antigen (T).

FIG. 21 shows the expected relative cell proliferation of CD4⁺ T cellsfrom a subject tolerant (A and C) or non-tolerant (B and D) to a donorantigen following culturing of the CD4⁺ T cells in the absence (A and B)or presence (C and D) of IL-5, IL-12, or IFN-γ and self antigen (S),donor antigen (D) or third party antigen (T).

DETAILED DESCRIPTION OF THE INVENTION

Tolerance is a critically important immunological discriminatory processwithout which a serious disease state might exist. As described above,there are circumstances when abnormal induction of an immune responseleads to autoimmune diseases, and the re-establishment of tolerancewould be desirable. There are also special circumstances when inductionof tolerance is desirable, for example, following organ transplantation.

There are also circumstances where induction of tolerance to a specificantigen is not desirable, for example, in the case of chronic infectionor cancer. In such circumstances, it would be desirable to reduce orbreak tolerance to the antigen of the infectious agent or tumour towhich a subject has developed tolerance to thereby reduce or eliminatethe chronic infection, or the tumour.

In one aspect, the invention relates to a method of assessing whether asubject comprises CD4⁺,CD25⁺ T cells that have been activated to aspecific antigen. The CD4⁺,CD25⁺ T cells activated to a specific antigenare capable of imparting tolerance to a specific antigen. Thus, theinvention also relates to a method of determining whether a subject istolerant, or capable of becoming tolerant, to a specific antigen. Asused herein, the term “tolerant” refers to the process of suppressing aportion of the immune system that recognises an antigen as beingforeign. It will be appreciated by persons skilled in the art that theterm “tolerance” as used herein has the same meaning as “immunetolerance”. Therefore, the phrase “tolerant to a specific antigen” willbe understood by those skilled in the art as meaning a state of immuneunresponsiveness or reduced immune responsiveness to the specificantigen without a prolonged generalised immunosuppression. Thus, asubject tolerant to a specific antigen is capable of eliciting an immuneresponse to antigens foreign to the subject other than the specificantigen. A subject that is “capable of becoming tolerant to a specificantigen” is one whose immune system contains the lymphocytes necessaryfor inducing tolerance to a specific antigen but which has not yetprogressed to full tolerance to the specific antigen.

The subject may be any subject which produces CD4⁺,CD25⁺ T cells. Formany years, animals of various species such as, for example mice andrats, have been used as models for studying the human immune system aswell as the immune system of other mammals. This has been the casebecause findings in mice and rats, for example, have been directlyapplicable to models of the immune system of humans and other mammals.Accordingly, results obtained in studies of mice, rats and other mammalsare directly applicable to humans and other mammals (Kostakis et al.IRCS Med Sci Libr Compend 1977, 5, 280).

The method of the invention for assessing whether a subject comprisesCD4⁺,CD25⁺ T cells that have been activated to a specific antigen,comprises obtaining from the subject a sample of lymphocytes comprisingCD4⁺,CD25⁺ T cells, incubating at least one portion of the sample oflymphocytes so as to promote distinction of CD4⁺,CD25⁺ T cells that havebeen activated to the specific antigen from CD4⁺,CD25⁺ T cells that havenot been activated to the specific antigen, and thereafter determiningwhether CD4⁺,CD25⁺ T cells that have been activated to the specificantigen are present in the sample.

The at least one portion of the sample may be incubated in any mannerthat promotes a distinction of CD4⁺,CD25⁺ T cells that have beenactivated to the specific antigen from other CD4⁺,CD25⁺ T cells. Thedistinction may, for example, be increased or decreased proliferation orsurvival of CD4⁺,CD25⁺ T cells activated to the specific antigenrelative to other CD4⁺,CD25⁺ T cells. This distinction may, for example,be increased or decreased expression of messenger RNA species by theCD4⁺,CD25⁺ T cells activated to the specific antigen relative to otherCD4⁺,CD25⁺ T cells.

Typically, the subject is a subject whose immune system is exposed tothe specific antigen. The term “lymphocyte” will be understood by thoseskilled in the art to refer to the cells of the immune system that areresponsible for initiating and controlling the specific immune response.Such cells include T lymphocytes, also known as T cells. CD4⁺lymphocytes are T lymphocytes. The sample of lymphocytes may be, forexample, mixed peripheral lymphocytes, isolated CD4⁺ lymphocytes orisolated CD4⁺,CD25⁺ lymphocytes. Typically, the sample of lymphocytes isperipheral blood lymphocytes.

The sample of lymphocytes is typically isolated from peripheral bloodlymphocytes. The isolation and characterisation of a population of Tlymphocyte cells in vitro has been described in a number of prior artdocuments, for example, those shown in U.S. Pat. No. 5,622,853 andInternational Patent Application No. WO00/20445; however, any knownprocedure for isolating lymphocytes may be used. Briefly, in oneoptional approach a blood sample containing T-cells is taken from amammal. Peripheral blood lymphocytes are then isolated from the bloodsample using the methods for T lymphocyte isolation referred to above.For example, peripheral blood lymphocytes may be isolated byFicoll-Hypaque gradient centrifugation (Pharmacia, Piscataway, N.J.).

Following isolation of peripheral blood lymphocytes, CD4⁺,CD25⁺ T cellsmay be isolated from the population of isolated peripheral bloodlymphocytei. As used herein, “CD4⁺,CD25⁺ T cell” is any lymphocyte thatexpresses on its surface the cluster of differentiation markers known asCD4 and CD25. A CD4⁺,CD25⁺ T cell is also known as CD4⁺, CD25⁺lymphocyte. The CD4⁺,CD25⁺ T cell may also express other markers whichmay aid in the isolation of CD4⁺,CD25⁺ T cells such as, for example,CD45RO⁻,RB⁻. Naïve CD4⁺,CD25⁺ T cells may express L-selectin. Typically,the CD4⁺,CD25⁺ T cells are CD4⁺,CD25^(+high) T cells.

Typically, CD4⁺,CD25⁺ T cells are isolated by positive enrichment ofCD25⁺ T cells using an anti-CD25 antibody. For example, CD4⁺,CD25⁺ Tcells may be isolated by means of multiparameter flow cytometricanalysis using one or more fluorescent labelled anti-CD25 antibodies.This method includes the analysis of both light scatter parameters aswell as one or more fluorescence parameters. Other methods of isolationinclude, for example, magnetic bead based separation as previouslydescribed in U.S. Pat. No. 517,101. Flow cytometric analysis may beperformed, for example, on a FACScan™ flow cytometer or a FACStar™ pluscell sorter (both available from Becton Dickinson ImmunocytometrySystems, “BDIS”). Data acquisition may be performed with FACScanResearch software and FACStar Plus software (BDIS). Forward lightscatter, orthogonal light scatter and three fluorescence signals aredetermined for each cell and stored in listmode data files. Eachexperiment measures approximately 30,000 cells, although it will beappreciated that the number of cells may vary greatly depending on thesubject and available lymphocytes. The analysis of the listmode datafiles is preferably performed with Paint-A-Gate, TM software (BDIS).(See U.S. Pat. No. 4,845,653). To increase the orthogonal lightscattering resolution, the orthogonal light scattering signals may betransformed by using a polynomial function as described in U.S. Pat.application Ser. No. 517,096. For light microscope examination, 10,000sorted cells are centrifuged for five minutes at 200 g and resuspendedin 100 ml RPMI 1640 containing 10% FCS. Cytospin preparations are madeon a Shandon cyto-centrifuge (Southern Products Ltd). Slides containingsorted cells may be stained with Wright Giemsa stain (Sigma).

CD4⁺,CD25⁺ T cells may be fluorescently labelled for identificationand/or isolation using a variety of monoclonal antibodies available fromBDIS. Antibodies may be fluorescent labelled with one of the followingfluorochromes: phycoerythrin (“PE”), fluorescein isothyocyanate (“FITC”)and peridinin chlorophyll complex (“PerCp”). For a description of PE andPerCp, see U.S. Pat. Nos. 4,520,110 and 4,876,190 respectively. Themonoclonal antibodies which may be used include, for example: anti-CD4FITC, PE or PerCp; anti-CD25 PE. (All antibodies commercially availablefrom BDIS).

CD4⁺,CD25⁻ T cells may be isolated by any methods known in the art. Forexample, CD4⁺,CD25′ T cells may be isolated by depleting CD4⁺ T cells ofCD4⁺,CD25⁺ T cells.

In some embodiments, the at least one portion of the sample oflymphocytes is contacted with the specific antigen prior to, orsimultaneously with, incubating the at least one portion of the samplein the absence of cytokines capable of stimulating activation ofCD4⁺,CD25⁺ T cells or of prolonging survival or supporting proliferationof activated CD4⁺,CD25⁺ T cells, or the presence of and one or morecytokines capable of prolonging survival or supporting proliferation ofactivated CD4⁺,CD25⁺ T cells. In some embodiments, the at least oneportion is contacted with the specific antigen by incubating in thepresence of specific antigen. The at least one portion of the sample maybe incubated in the presence of the specific antigen in any manner thatpermits lymphocytes in the sample to contact the specific antigen and torecognise the specific antigen. As used herein, the term “contacting” or“contacted” refers to contacting a lymphocyte with an antigen in amanner which permits the lymphocyte to recognise the antigen. In otherwords, the at least one portion of the sample of lymphocytes arecontacted with the specific antigen in a manner which would permitactivation of T cells in the sample.

Typically, the at least one portion of the sample of lymphocytes iscontacted with the specific antigen by presenting the specific antigento the lymphocytes on the surface of a stimulator cell such as, forexample, an antigen presenting cell. Typically, the specific antigen ispresented to the lymphocytes associated with a major histocompatability(MHC) molecule (typically class II) on the surface of an antigenpresenting cell. As defined herein, a “stimulator cell” is a cell whichis capable of presenting an antigen to a lymphocyte in a manner in whichthe lymphocyte can recognise the antigen. For example, the stimulatorcell may be a tumour cell (see for example U.S. Pat. No. 5,342,774,Knuth et al. (Proc. Natl. Acad. Sci. USA 86: 2804-2808, 1989) and VanDen Eynde et al. (Int. J. Cancer 44: 634-640, 1989) or the stimulatorcell may be an antigen presenting cell.

An “antigen presenting cell” will be understood by those skilled in theart to be a cell which contributes to the induction of an immuneresponse by presenting antigen to T-lymphocytes. Antigen presentingcells may be dendritic cells, mononuclear phagocytes, B-lymphocytes,unfractionated lymphocytes or Langerhans cells. The antigen presentingcells may be isolated from, for example, bone marrow, blood, thymus,epidermis, liver or fetal liver. The antigen presenting cells may beunfractionated lymphocytes in which stimulator cells have been impairedby treatment with, for example, irradiation or mitomycin C. The antigenpresenting cells may be cells expressing the relevant antigen presentingmolecule (eg. Class II MHC) and other ligands that are required tofacilitate binding and activation of naïve CD4⁺,CD25⁺ T cells. Suitableligands include ICAM1, ICAM2, LFA3, and the ligands for CD28 and CTL-Aand other activation ligands or part of the antigen that is presented onself MHC molecules and recognised by T cells activated in an autoimmuneresponse.

The at least one portion of the sample of lymphocytes may be contactedwith the specific antigen using synthetic antigen presenting systems,such as those described in U.S. Pat. No. 6,828,150 or 6,787,154.

As will be apparent to those skilled in the art, a sample of lymphocytesmay be contacted with other antigens by the same manner as describedabove for contacting the at least one portion of the sample oflymphocytes with the specific antigen.

The specific antigen may be any substance which elicits an immuneresponse in a subject that is not tolerant to the specific antigen. Thespecific antigen may or may not be derived from the subject. Thespecific antigen may be an autoantigen, which will be understood bythose skilled in the art as referring to an antigen that can elicit areaction in persons with a propensity to allergy. The specific antigenmay be an alloantigen, which will be understood by those skilled in theart as referring to an antigen derived from a subject of the samespecies. The specific antigen may be a xenoantigen, which will beunderstood by those skilled in the art as referring to an antigenderived from a subject of a different species. The specific antigen maybe an allergen.

As mentioned above, the specific antigen may be any substance whichelicits an immune response in a subject that is not tolerant to theantigen. For example, a typical alloantigen may be donor transplantcells or tissue from another human. A typical xenoantigen may betransplant cells or tissue from a non-human animal such as, for example,a pig. Donor transplant cells or tissue from humans or non-human animalsmay include kidney, liver, heart, lung, skin, pancreas, cornea, lens,bone marrow, muscle, connective tissue, vascular tissue,gastrointestinal tissue, nervous tissue, bone, valves, stem cells,cells, such as stem cells, transfected with an agent such as atherapeutic agent.

The antigen presenting cell may be isolated with the specific antigenalready presented on the surface of the cell. For example, antigenpresenting cells isolated from, for example, the spleen of a subjectsuffering from an autoimmune disease will have the autoantigen presentedon the surface of the cell. In the case of tissue transplantation,antigen presenting cells isolated from the tissue of a transplant donorwill have the alloantigen presented on the surface of such cells. Forexample, the antigen presenting cells may be frozen or stored spleen orlymph node cells from the cadaver of a donor, or peripheral blood cellsfrom a living donor. Alternatively, empty MHC molecules of antigenpresenting cells isolated from the subject may be loaded with specificantigens as described in U.S. Pat. No. 5,731,160 whereby empty MHCmolecules are loaded with immunogenic exogenous peptides ofapproximately 8 to 18 amino acids in length.

Antigen presenting cells may be isolated from blood or tissue by methodsknown in the art. For example, B-lymphocytes can be purified from amixed population of cells (e.g. other cell types in peripheral blood orspleen) by standard cell separation techniques. For example, adherentcells can be removed by culturing spleen cells on plastic dishes andrecovering the non-adherent cell population. T-lymphocytes can beremoved from a mixed population of cells treated with an anti-T cellantibody (e.g. anti-CD3 (see for example WO 01/37860), anti-CD2) andcomplement. In one embodiment, resting B-lymphocytes are used as theantigen presenting cell. Resting B-lymphocytes can be isolated bymethods based on the small size and density of the B-lymphocytes.Resting lymphoid cells may be isolated by counterflow centrifugalelutriation as described in Tony, H-P, Parker, D.C. (1985) J. Exp. Med.161: 223-241. Using counterflow centrifugal elutriation, a small,resting lymphoid cell population depleted of cells which can activate Tcell responses can be obtained as described in U.S. Pat. No. 6,312,692.

In another embodiment, unfractionated lymphocytes may be used as theantigen presenting cell. Typically, the unfractionated lymphocytes aretreated to impair proliferation of stimulator cells. Examples oftreatments suitable for impairing proliferation of stimulator cellsinclude irradiation, or treatment with mitomycin C.

In one embodiment of the method of the first aspect of the presentinvention, simultaneous with, or following contacting the at least oneportion of the sample of lymphocytes with the specific antigen, the atleast one portion of the sample of lymphocytes is incubated:

-   -   (i) in the absence of cytokines capable of stimulating        activation of CD4⁺,CD25⁺ T cells or of prolonging survival or        supporting proliferation of activated CD4⁺,CD25⁺ T cells; or    -   (ii) in the presence of at least one cytokine selected from the        group consisting of IL-5, IL-12, IL-23 and IFN-γ, or a        biologically active fragment thereof, or a functionally        equivalent molecule thereof.

In some embodiments, a portion of the sample is contacted with thespecific antigen and incubated in the absence of cytokines capable ofstimulating activation of CD4⁺,CD25⁺ T cells or of prolonging survivalor supporting proliferation of activated CD4⁺,CD25⁺ T cells, and anotherportion of the sample is contacted with the specific antigen andincubated in the presence of at least one cytokine selected from thegroup consisting of IL-5, IL-12, IL-23 and IFN-γ, or abiologicallyactive fragment thereof, or a functionally equivalent molecule thereof.

It will be understood by those skilled in the art that the expression“in the absence of cytokines capable of stimulating activation ofCD4⁺,CD25⁺ T cells or of prolonging survival or supporting proliferationof activated CD4⁺,CD25⁺ T cells” means that those cytokines that arecapable of stimulating activation of CD4⁺,CD25⁺ T cells (such as IL-2 orIL-4, or a biologically active fragment thereof, or a functionallyequivalent molecule thereof) or of prolonging survival or supportingproliferation of activated CD4⁺,CD25⁺ T cells (such as IL-5, IL-12,IL-23 and IFN-γ, or a biologically active fragment thereof, or afunctionally equivalent molecule thereof), are either not present, orare present in such low amounts that they do not have a biologicaleffect. As used herein, the expression “biological effect” refers to theability to activate CD4⁺,CD25⁺ T cells or to prolong survival or supportproliferation of activated CD4⁺,CD25⁺ T cells. It will also beunderstood that the expression “in the presence of at least one cytokineselected from the group consisting of IL-5, IL-12, IL-23 or IFN-γ, or abiologically active fragment thereof, or a functionally equivalentmolecules thereof” means that one or more of IL-5, IL-12, IL-23 orIFN-γ, or a biologically active fragment thereof, or a functionallyequivalent molecule thereof, is present in a sufficient amount tomaintain survival and/or promote proliferation of activated CD4⁺,CD25⁺ Tcells. The expression “biologically active fragment thereof” in relationto IL-5, IL-12, IL-23 or IFN-γ refers to any fragment of these cytokineswhich has the ability to maintain survival or support proliferation ofCD4⁺,CD25⁺ T cells activated to a specific antigen, and in relation toIL-2 and IL-4 refers to any fragment of these cytokines which has theability to support activation of naïve CD4⁺,CD25⁺ T cells. For example,a biologically active fragment of IL-5 may be any portion of the IL-5molecule which has the ability to maintain survival and/or supportproliferation of CD4⁺,CD25⁺ T cells activated to a specific antigen.

It is envisaged by the inventors that as the cytokines IL-5, IL-12,IL-23 and/or IFN-γ are capable of prolonging survival, or supportingproliferation, of activated CD4⁺,CD25⁺ T cells, any molecules whichinteract with the IL-5, IL-12, IL-23 or IFN-γ receptor of the activatedCD4⁺,CD25⁺ T cells to activate the same signal transduction pathways asIL-5, IL-12, IL-23 or IFN-γ will also be effective in prolongingsurvival and/or supporting proliferation of activated CD4⁺,CD25⁺ Tcells. As used herein, the term “functionally equivalent molecule”refers to a molecule that is not a cytokine or a biologically activefragment thereof, but which has the same biological activity as acytokine. Accordingly, the expression “functionally equivalent moleculethereof” in relation to IL-5, IL-12, IL-23 or IFN-γ refers to a moleculethat is not IL-5, IL-12, IL-23 or IFN-1, or a biologically activefragment thereof, but which is nonetheless a ligand for the IL-5, IL-12,IL-23 or IFN-γ receptor and which is capable of prolonging survivaland/or supporting proliferation of CD4⁺,CD25⁺ T cells activated to aspecific antigen. As used herein (other than in this paragraph), theterm “cytokine” includes biologically active fragments of cytokines andfunctionally equivalent molecules of cytokines.

The at least one portion of the sample of lymphocytes may be incubatedin a medium. Suitably, the medium is cell culture medium. Lymphocytesincubated in the absence of cytokines capable of stimulating activationof CD4⁺,CD25⁺ T cells or of prolonging survival or supportingproliferation of activated CD4⁺,CD25⁺ T cells will typically beincubated in medium that does not contain cytokines, or containscytokines in such low amounts that they do not stimulate activation ofCD4⁺,CD25⁺ T cells or prolong survival or support proliferation ofactivated CD4⁺,CD25⁺ T cells. Cytokine free medium is known in the artand is that with no foreign serum, conditioned media or specificcytokines has been added. The at least one portion of the sample oflymphocytes incubated in the presence of at least one cytokine selectedfrom the group consisting of IL-5, IL-12, IL-23 and IFN-γ, or abiologically active fragment thereof, or a functionally equivalentmolecule thereof, may be incubated in the same media as mentioned above,to which has been added the at least one cytokine selected from thegroup consisting of IL-5, IL-12, IL-23 and IFN-γ, or a biologicallyactive fragment thereof, or a functionally equivalent molecule thereof.

Activated CD4⁺,CD25⁺ T cells are typically capable of conferringtolerance in a subject to the antigen to which the CD4⁺,CD25⁺ T cellshave been activated. Thus, it would be expected that the peripheralblood of a subject that was tolerant to a specific antigen wouldcomprise CD4⁺,CD25⁺ T cells activated to the specific antigen that arecapable of conferring tolerance to that specific antigen. However, asdiscussed above, the inventors have found that CD4⁺,CD25⁺ T cellsactivated to a specific antigen are short lived in the absence ofspecific cytokines and the activated CD4⁺,CD25⁺ T cells are generallynot viable or have reduced viability after a period of between 24 hoursand 1 week following contact with the specific antigen, unless theactivated CD4⁺,CD25⁺ T cells are incubated in the presence of at leastone cytokine selected from the group consisting of IL-5, IL-12, IL-23and IFN-γ, or a biologically active fragment thereof, or a functionallyequivalent molecule thereof.

The inventors have developed methods whereby a sample of lymphocytes maybe incubated so as to promote a distinction between CD4⁺,CD25⁺ T cellsthat have been activated to a specific antigen from those that have notbeen activated to the specific antigen. For example, the inventors haveobserved that CD4⁺,CD25⁺ T cells activated to a specific antigen are notcapable of proliferation, and typically die, when contacted withspecific antigen in media which does not contain cytokines capable ofprolonging survival or supporting proliferation of activated CD4⁺,CD25⁺T cells. Accordingly, CD4⁺,CD25⁺ T cells activated to the specificantigen will not proliferate in the absence of cytokines which arecapable of stimulating activation of CD4⁺,CD25⁺ T cells or of prolongingsurvival or supporting proliferation of activated CD4⁺,CD25⁺ T cells,such as IL-5, IL-12, IL-23 and IFN-γ, or a biologically active fragmentthereof, or a functionally equivalent molecules thereof, and toleranceto a specific antigen can therefore be determined by low or noproliferation of CD4⁺,CD25⁺ T cells when the lymphocytes are contactedwith a specific antigen in the absence of cytokines capable ofstimulating activation of activated CD4⁺,CD25⁺ T cells or of prolongingsurvival or supporting proliferation of CD4⁺,CD25⁺ T cells.

The presence of CD4⁺,CD25⁺ T cells activated to the specific antigen maybe detected by, for example, measuring the proliferation of CD4⁺,CD25⁺ Tcells in the absence of cytokines which are capable of stimulatingactivation of CD4⁺,CD25⁺ T cells or prolonging survival or supportingproliferation of activated CD4⁺,CD25⁺ T cells, relative to that ofCD4⁺,CD25⁺ T cells in the presence of at least one cytokine selectedfrom the group consisting of IL-5, IL-12, IL-23 and IFN-γ, or abiologically active fragment thereof, or a functionally equivalentmolecule thereof. Thus, in one embodiment, tolerance to a specificantigen in a subject may be determined by:

-   -   (a) obtaining from the subject a sample comprising CD4⁺,CD25⁺ T        cells;    -   (b) contacting the CD4⁺,CD25⁺ T cells with the specific antigen;    -   (c) incubating a first portion of the CD4⁺,CD25⁺ T cells in the        absence of cytokines capable of stimulating activation of        CD4⁺,CD25⁺ T cells or of prolonging survival or supporting        proliferation of activated CD4⁺,CD25⁺ T cells, and a second        portion of the CD4⁺,CD25⁺ T cells in the presence of at least        one cytokine selected from the group consisting of IL-5, IL-12,        IL-23 and IFN-γ, or a biologically active fragment thereof, or a        functionally equivalent molecule thereof;    -   (d) thereafter detecting whether the CD4⁺,CD25⁺ T cells from        each portion have proliferated whereby tolerance to the specific        antigen, is indicated by increased proliferation of the        CD4⁺,CD25⁺ T cells of the second portion relative to the        CD4⁺,CD25⁺ T cells of the first portion.

Lack of tolerance of the subject for the specific antigen is indicatedby a decrease in proliferation, or no change in proliferation, of theCD4⁺,CD25⁺ T cells of the second portion relative to the first portion.

In one form, CD4⁺ T cells may be used in combination with an antibodywhich removes or inactivates CD4⁺,CD25⁻ T cells such as, for example,anti-CD45RB RO. Thus, in another embodiment, tolerance to a specificantigen in a subject may be determined by:

-   -   (a) obtaining from the subject a sample comprising CD4⁺ T cells;    -   (b) contacting the CD4⁺ T cells with the specific antigen;    -   (c) incubating the CD4⁺ T cells in the presence of an antibody        which inactivates CD4⁺,CD25⁻ T cells;    -   (d) incubating a first portion of the CD4⁺ T cells in the        absence of cytokines capable of stimulating activation of        CD4⁺,CD25⁺ T cells or of prolonging survival or of supporting        proliferation of CD4⁺CD25⁺ T cells, and a second portion of the        CD4⁺ T cells in the presence of at least one cytokine selected        from the group consisting of IL-5, IL-12, IL-23 and IFN-γ, or a        biologically active fragment thereof, or a functionally        equivalent molecule thereof;    -   (e) thereafter detecting whether CD4⁺,CD25⁺ T cells from each        portion have proliferated whereby tolerance to the specific        antigen is indicated by proliferation of the CD4⁺,CD25⁺ T cells        of the second portion relative to the T cells of the first        portion.

Lack of tolerance of the subject for the specific antigen is indicatedby a decrease, or no change, in proliferation of the CD4⁺,CD25⁺ T cellsof the second portion relative to the first portion.

Typically the antibody which inactivates CD4⁺,CD25⁻ T cells is ananti-CD45 RB/RO antibody, such as that described in WO02/072832.

As CD4⁺,CD25⁺ T cells activated to a specific antigen will notproliferate unless they are incubated in the presence of the specificantigen and cytokines which support proliferation of activatedCD4⁺,CD25⁺ T cells, in samples obtained from a subject which hasCD4⁺,CD25⁺ T cells activated to the specific antigen, the first portionwill not proliferate, while the second portion, which is incubated inthe presence of at least one cytokine selected from the group consistingof IL-5, IL-12, IL-23 and IFN-γ, or a biologically active fragmentthereof, or a functionally equivalent molecule thereof, willproliferate.

Optionally, the lymphocytes are incubated in the presence of anti-IL-2and/or anti-IL-4 antibody to inactivate any IL-2 or IL-4.

Alternatively, proliferation of CD4⁺,CD25⁺ T cells may be determined inCD4⁺ T cell populations by measuring the proliferation of CD4⁺,CD25⁻ Tcells. As CD4⁺,CD25⁺ T cells activated to a specific antigen are notcapable of growth or survival in the presence of the specific antigenand in the absence of cytokines which are capable of stimulatingactivation of CD4⁺,CD25⁺ T cells or of prolonging survival or supportingproliferation of activated CD4⁺,CD25⁺ T cells, they are therefore notcapable of inhibiting activation and proliferation of the CD4⁺,CD25⁻population in response to the specific antigen in the absence ofcytokines which are capable of stimulating activation of CD4⁺,CD25⁺ Tcells or of prolonging survival or stimulating proliferation ofactivated CD4⁺,CD25⁺ T cells. However, a portion of CD4⁺,CD25⁺ T cellswill proliferate in response to third party antigen, and will thereforesuppress CD4⁺,CD25⁻ T cells in response to the third party antigen.

Accordingly, tolerance may be determined in a sample of CD4⁺ lymphocytesfrom a subject by comparing proliferation of unfractionated CD4⁺ T cellsto that of CD4⁺,CD25⁻ T cells. In the absence of cytokines capable ofstimulating activation of CD4⁺,CD25⁺ T cells, proliferation ofCD4⁺,CD25⁻ T cells in response to third party antigen will increaserelative to that of the mixed population in CD4⁺ T cells from a subjecttolerant to a specific antigen. In contrast, in the absence of cytokinescapable of stimulating activation or prolonging survival or supportingproliferation of CD4⁺,CD25⁺ T cells, proliferation of isolatedCD4⁺,CD25⁻ T cells in response to specific antigen will not increaserelative to that of the mixed population in CD4⁺ T cells from a subjecttolerant to a specific antigen. Thus, in another embodiment, the methodcomprises the steps of:

-   -   (a) obtaining from the subject a sample of lymphocytes        comprising CD4⁺ T cells;    -   (b) preparing from the sample a CD4⁺ T cell population and a        CD4⁺,CD25⁻ T cell population;    -   (c) contacting a first portion of the CD4⁺ T cell population        with the specific antigen, and a first portion of the CD4⁺,CD25⁻        T cell population with the specific antigen, and contacting a        second portion of the CD4⁺ T cell population with a further        antigen, and contacting a second portion of the CD4⁺,CD25⁻ T        cell population with a further antigen;    -   (d) incubating the first and second portions in the absence of        cytokines that are capable of stimulating activation of        CD4⁺,CD25⁺ T cells or of prolonging survival or stimulating        proliferation of activated CD4⁺CD25⁺ T cells; and    -   (e) thereafter comparing proliferation of the portions of the        CD4⁺ T cell population and the CD4⁺,CD25⁻ T cell population in        the first and second portions, whereby low or no increase in        proliferation of the CD4⁺,CD25⁻ T cell population relative to        that of the CD4⁺ T cell population in the first portions, and        increased proliferation of the CD4⁺,CD25⁻ T cell population        relative to the CD4⁺ T cell population in the second portions,        is indicative of tolerance to the specific antigen in the        subject.

Typically, the CD4⁺,CD25⁻ T cell population is prepared by depletingCD4⁺,CD25⁺ T cells from a portion of the sample of lymphocyte comprisingCD4⁺ T cells.

In this embodiment, CD4⁺,CD25⁺ T cells activated to the specific antigenin the first portion from a subject tolerant to the specific antigenwill not proliferate in response to the specific antigen and will not beable to suppress proliferation of CD4⁺,CD25⁻ T cell population in theunfractionated CD4⁺ T cells. Thus, proliferation of the CD4⁺ T cellswill be similar to proliferation of the first portion of CD4⁺,CD25⁻ Tcells following contacting the T cells with specific antigen. However,CD4⁺,CD25⁺ T cells in the CD4⁺ population of the second portion willproliferate following contact with a further antigen and will thereforebe capable of inhibiting proliferation of CD4⁺,CD25⁻ lymphocytes inunfractionated CD4⁺ T cells. When the CD4⁺,CD25⁺ T cells are removedfrom the CD4⁺ T cell population, the resulting CD4⁺,CD25⁻ T cells areenriched, and these enriched cells of the second portion exhibit greaterproliferation following contact with the further antigen.

If the subject does not comprise CD4⁺,CD25⁺ T cells activated to thespecific antigen, then the level of proliferation in response to thespecific antigen in the first portion will be similar to proliferationfollowing contact with the further antigen in the second portion, thatis the response of the CD4⁺,CD25⁻ T cell population will be greater thanthat of unfractionated CD4⁺ T cells in the first portion.

It is also possible to determine tolerance by detecting proliferation ofCD4⁺,CD25⁺ T cells activated to the specific antigen in the presence ofIL-5, IL-12, IL-23 or IFN-γ, or a biologically active fragment thereof,or a functionally equivalent molecule thereof, and in the absence ofcytokines which are capable of stimulating activation of CD4⁺,CD25⁺ Tcells, because naïve CD4⁺,CD25⁺ T cells are not capable of proliferationin the presence of any one or more of IL-5, IL-12, IL-23 or IFN-γ, or abiologically active fragment thereof, or a functionally equivalentmolecule thereof, and in the absence of cytokines which are capable ofstimulating activation of CD4⁺,CD25⁺ T cells. Thus, in the presence ofIL-5, IL-12, IL-23 or IFN-γ, or a biologically active fragment thereof,or a functionally equivalent molecule thereof, and in the absence ofcytokines which are capable of stimulating activation of CD4⁺,CD25⁺ Tcells, only activated CD4⁺,CD25⁺ T cells will proliferate.

Proliferation of CD4⁺,CD25 T cells activated to a specific antigen willinhibit proliferation of CD4⁺,CD25⁻ T cells in response to the specificantigen. Thus, proliferation of CD4⁺,CD25⁺ T cells activated to thespecific antigen in the presence of IL-5, IL-12, IL-23 and/or IFN-γ, orbiologically active fragments thereof, or functionally equivalentmolecules thereof, may be detected by measuring the proliferation ofunfractionated CD4⁺ T cells in the presence of at least one cytokineselected from the group consisting of IL-5, IL-12, IL-23 and IFN-γ, or abiologically active fragment thereof, or a functionally equivalentmolecule thereof, and in the absence of cytokines which are capable ofstimulating activation of CD4⁺,CD25⁺ T cells. Accordingly, in anotherembodiment, the method comprises the steps of:

-   (a) obtaining from the subject a sample comprising unfractionated    lymphocytes, the sample comprising CD4⁺ T cells;-   (b) contacting the unfractionated lymphocytes with the specific    antigen;-   (c) incubating a first portion of the unfractionated lymphocytes in    the absence of cytokines which are capable of stimulating activation    of CD4⁺,CD25⁺ T cells or of prolonging survival or supporting    proliferation of activated CD4⁺,CD25⁺ T cells, and a second portion    of the unfractionated lymphocytes in the presence of at least one    cytokine selected from the group consisting of IL-5, IL-12, IL-23    and IFN-γ, or a biologically active fragment thereof, or a    functionally equivalent molecule thereof;-   (d) thereafter detecting proliferation of unfractionated lymphocytes    in the first portion relative to the second portion, whereby reduced    proliferation of CD4⁺ T cells in the second portion relative to the    first portion indicates that the subject is tolerant to the specific    antigen.

In unfractionated lymphocytes populations, proliferation of CD4⁺,CD25⁺ Tcells activated to the specific antigen in the presence of at least onecytokine selected from the group consisting of IL-5, IL-12, IL-23 andIFN-γ, or a biologically active fragment thereof, or a functionallyequivalent molecule thereof, results in inhibition of proliferation ofCD4⁺,CD25⁻ T cells in response to the specific antigen. Theproliferation of CD4⁺,CD25⁻ T cells is usually an order of magnitudegreater than that of the minority CD4⁺,CD25⁺ T cells, thus theinhibition of the CD4⁺,CD25⁻ population is not exceeded by the increasedproliferation of the minor CD4⁺,CD25⁺ T cell population. The net effectin a sample from a tolerant subject is inhibition of proliferation ofthe unfractionated CD4⁺ T cells incubated in the presence of at leastone cytokine selected from the group consisting of IL-5, IL-12, IL-23and IFN-γ, or a biologically active fragment thereof, or a functionallyequivalent molecule thereof, relative to proliferation of unfractionatedCD4⁺ T cells incubated in the absence of cytokines capable ofstimulating activation of CD4⁺,CD25⁺ T cells or of prolonging survivalor supporting proliferation of activated CD4⁺,CD25⁺ T cells. The lack ofproliferation of CD4⁺,CD25⁺ T cells in the absence of cytokines capableof stimulating activation of CD4⁺,CD25⁺ T cells or of prolongingsurvival or supporting proliferation of activated CD4⁺,CD25⁺ T cells,results in proliferation of CD4⁺,CD25⁻ T cells (in the CD4⁺ population)in response to the specific antigen as this response is uninhibited byCD4⁺,CD25⁺ T cells activated to the specific antigen which die withoutthe specified cytokines.

The inventors have further found that while CD4⁺,CD25⁺ T cells activatedto a specific antigen are not capable of proliferation in the absence ofcytokines capable of stimulating activation of CD4⁺,CD25⁺ T cells, or ofprolonging survival or supporting proliferation of activated CD4⁺,CD24⁺T cells, following contact with the specific antigen, the activatedCD4⁺,CD25⁺ T cells will proliferate in response to contact with afurther antigen in the absence of cytokines capable of stimulatingactivation of CD4⁺,CD25⁺ T cells, or of prolonging survival orsupporting proliferation of activated CD4⁺,CD25⁺ T cells. Accordingly,in another embodiment, the method comprises the steps of:

-   (a) obtaining from the subject a sample comprising CD4⁺,CD25⁺ T    cells;-   (b) contacting a first portion of the CD4⁺,CD25⁺ T cells with the    specific antigen, and a second portion of the CD4⁺,CD25⁺ T cells    with a further antigen;-   (c) incubating the first and second portions in the absence of    cytokines which are capable of stimulating activation of CD4⁺,CD25⁺    T cells or prolonging survival or supporting proliferation of    activated CD4⁺,CD25⁺ T cells;-   (d) thereafter detecting the proliferation of CD4⁺,CD25⁺ T cells in    the first and second portion, whereby greater proliferation of    CD4⁺,CD25⁺ T cells in the second portion relative to CD4⁺,CD25⁺ T    cells in the first portion indicates that the subject is tolerant to    the specific antigen.

It will be understood by those skilled in the art that the furtherantigen is different to that of the specific antigen. For example, wherethe specific antigen may be a donor antigen, the further antigen may bea third party antigen.

The inventors have further found that proliferation of CD4⁺,CD25⁻ Tcells from animals tolerant to a specific antigen is not fullysuppressed by CD4⁺,CD25⁺ T cells from tolerant animals when admixed at aratio of CD4⁺,CD25⁺ T cells: CD4⁺,CD25⁻ T cells of greater than about1:3, typically about 1:1, when incubated in the absence of cytokinescapable of stimulating activation of CD4⁺,CD25⁺ T cells, or ofprolonging survival or supporting proliferation of CD4⁺,CD25⁺ T cellsactivated to the specific antigen. This contrasts with the ability ofnaïve CD4⁺,CD25⁺ T cells to fully inhibit the responses of naïveCD4⁺,CD25⁻ T cells when present in a ratio of at least 1:3, typically1:1. As IL-5, IL-12, IL-23 or IFN-γ can prolong survival and supportproliferation of CD4⁺,CD25⁺ T cells activated to a specific antigen,incubation of CD4⁺,CD25⁺ T cells with CD4⁺,CD25⁻ T cells from a subjecttolerant to a specific antigen in the presence of specific antigen andone or more of IL-5, IL-12, IL-23 or IFN-γ, a biologically activefragment thereof, or a functionally equivalent molecule thereof, wouldresult in inhibition of proliferation of the CD4⁺,CD25⁻ T cells.

Accordingly, in another embodiment, the method comprises the steps of:

-   (a) obtaining from the subject a sample comprising CD4⁺,CD25⁺ T    cells and CD4⁺,CD25⁻ T cells;-   (b) preparing from the sample an admixed T cell population    comprising CD4⁺,CD25⁺ T cells and CD4⁺,CD25⁻ T cells in a ratio of    greater than about 1:3 (CD4⁺,CD25⁺ T cells:CD4⁺,CD25⁻ T cells),    typically about 1:1;-   (c) contacting a first portion of the admixed T cell population with    the specific antigen, and a second portion of the admixed T cell    population with a further antigen;-   (d) incubating the first and second portions in the absence of    cytokines which are capable of stimulating activation of CD4⁺,CD25⁺    T cells or of prolonging survival or supporting proliferation of    activated CD4⁺,CD25⁺ T cells;-   (e) thereafter determining proliferation of the first portion    relative to the second portion whereby greater proliferation in the    first portion relative to the second portion indicates that the    subject is tolerant to the specific antigen.

Typically, a CD4⁺,CD25⁻ T cell population is prepared by depletingCD4⁺,CD25⁺ T cells from a portion of the sample, and a CD4⁺,CD25⁺ T cellpopulation is prepared by depleting CD4⁺,CD25⁻ T cells from a portion ofthe sample, and the admixed T cell population prepared by mixing theCD4⁺,CD25⁺ T cell population and the Cpe,CD25⁻ T cell population toobtain the ratio of CD4⁺,CD25⁺ T cells:CD4⁺,CD25⁻ T cells of greaterthan 1:3.

As the first and second portions are incubated in the absence ofcytokines capable of stimulating activation of CD4⁺,CD25⁺ T cells or ofprolonging survival or supporting proliferation of CD4⁺,CD25⁺ T cellsactivated to the specific antigen, then CD4⁺,CD25⁺ T cells activated tothe specific antigen will not survive or proliferate in response to thespecific antigen and will therefore not be able to inhibit proliferationof the CD4⁺,CD25⁻ T cells. In contrast, naïve CD4⁺,CD25⁺ T cells will beable to inhibit proliferation of CD4⁺,CD25⁻ T cells in the secondportion.

In another embodiment, the method comprises the steps of:

-   (a) obtaining from the subject a sample comprising CD4⁺,CD25⁺ T    cells and CD4⁺,CD25⁻ T cells;-   (b) preparing from the sample a CD4⁺CD25⁻ T cell population, and an    admixed T cell population comprising CD4⁺,CD25⁺ T cells and    CD4⁺,CD25⁻ T cells in a ratio of greater than 1:3;-   (c) contacting the CD4⁺,CD25⁻ T cell population and the admixed T    cell population with the specific antigen;-   (d) incubating the CD4⁺,CD25⁻ T cell population and the admixed T    cell population in the absence of cytokines which are capable of    stimulating activation of CD4⁺,CD25⁺ T cells or of prolonging    survival or supporting proliferation of activated CD4⁺,CD25⁺ T    cells;-   (e) thereafter determining proliferation of the CD4⁺,CD25⁻ T cell    population and the admixed T cell population whereby proliferation    of the admixed T cell population substantially the same as that of    the CD4⁺,CD25⁻ T cell population indicates that the subject is    tolerant to the specific antigen.

Typically, a CD4⁺,CD25⁻ T cell population is prepared by depletingCD4⁺,CD25⁺ T cells from a portion of the sample, and a CD4⁺,CD25⁺ T cellpopulation is prepared by depleting CD4⁺,CD25⁻ T cells from a portion ofthe sample, and the admixed T cell population prepared by mixing theCD4⁺,CD25⁺ T cell population and the CD4⁺,CD25⁻ T cell population toobtain the ratio of CD4⁺,CD25⁺ T cells:CD4⁺,CD25⁻ T cells of greaterthan 1:3.

In this embodiment, in the absence of cytokines capable of stimulatingactivation of CD4⁺,CD25⁺ T cells or of prolonging survival or supportingproliferation of activated CD4⁺,CD25⁺ T cells, activated CD4⁺,CD25⁺ Tcells in the admixed T cell population would not survive and wouldtherefore not be able to suppress proliferation of the CD4⁺,CD25⁻ Tcells. The effect would be that proliferation of the CD4⁺,CD25⁻ T cellsin the admixed T cell population would be approximately the same as thatof the CD4⁺,CD25⁻ T cell population.

In another embodiment, the method comprises the steps of:

-   (a) obtaining from the subject a sample comprising CD4⁺,CD25⁺ T    cells and CD4⁺,CD25⁻ T cells;-   (b) preparing from the sample an admixed T cell population by mixing    the CD4⁺,CD25⁺ T cells and the CD4⁺,CD25⁻ T cells in a ratio greater    than 1:3, typically 1:1;-   (c) contacting the admixed T cell population with the specific    antigen;-   (d) incubating a first portion of the admixed T cell population in    the absence of cytokines capable of stimulating activation of    CD4⁺,CD25⁺ T cells or of prolonging survival or supporting    proliferation of activated CD4⁺CD25⁺ T cells, and a second portion    of the admixed T cell population in the presence of at least one    cytokine selected from the group consisting of IL-5, IL-12, IL-23    and IFN-γ, or a biologically active fragment thereof, or a    functionally equivalent molecule thereof;-   (e) thereafter determining proliferation of T cells in the first and    second portions whereby greater proliferation of T cells in the    first portion relative to the second portion indicates tolerance to    the specific antigen.

Typically, a CD4⁺,CD25⁻ T cell population is prepared by depletingCD4⁺,CD25⁺ T cells from a portion of the sample, and a CD4⁺,CD25⁺ T cellpopulation is prepared by depleting CD4⁺,CD25⁻ T cells from a portion ofthe sample, and the admixed T cell population prepared by mixing theCD4⁺,CD25⁺ T cell population and the CD4⁺,CD25⁻ T cell population toobtain the ratio of CD4⁺,CD25⁺ T cells:CD4⁺,CD25⁻ T cells of greaterthan 1:3.

In this embodiment, CD4⁺,CD25⁺ T cells activated to the specific antigensurvive and proliferate in the presence of the at least one Cytokineselected from the group consisting of IL-5, IL-12, IL-23 and IFN-γ, or abiologically active fragment thereof, or a functionally equivalentmolecule thereof, and as a consequence, inhibit proliferation of theCD4⁺,CD25⁻ T cells. In contrast, in the absence of cytokines capable ofstimulating activation of CD4⁺,CD25⁺ T cells or of prolonging survivalor supporting proliferation of activated CD4⁺,CD25⁺ T cells, CD4⁺,CD25⁺T cells activated to the specific antigen do not proliferate or surviveand therefore do not inhibit CD4⁺,CD25⁻ T cells. As a consequence, inthe presence of CD4⁺,CD25⁺ T cells activated to the specific antigen,proliferation of T cells is greater in the first portion.

In embodiments where the at least one portion of the sample oflymphocytes is incubated in the presence of at least one cytokineselected from the group consisting of IL-5, IL-12, IL-23 and IFN-γ, or abiologically active fragment thereof, or a functionally equivalentmolecule thereof, the at least one sample of lymphocytes may, in someembodiments, be incubated in the presence of:

-   (a) at least one cytokine selected from the group consisting of    IL-5, IL-12 and IFN-γ, or a biologically active fragment thereof, or    a functionally equivalent molecule thereof; or-   (b) IL-5, a biologically active fragment thereof, or a functionally    equivalent molecule thereof; or-   (c) IL-12, a biologically active fragment thereof, or functionally    equivalent molecule thereof; or-   (d) IFN-γ, a biologically active fragment thereof, or a functionally    equivalent molecule thereof; or-   (e) IL-23, a biologically active fragment thereof, or a functionally    equivalent molecule thereof.

The period of time in which the at least one portion of the sample oflymphocytes is incubated to promote distinction of CD4⁺,CD25⁺ T cellsthat have been activated to a specific antigen from CD4⁺,CD25⁺ T cellsthat have not been activated to the specific antigen may include: 24hours to 10 days; 24 hours to 9 days; 24 hours to 8 days; 24 hours to 7days; 24 hours to 6 days; 24 hours to 5 days; 24 hours to 4 days; 24hours to 3 days.

As used herein, the term “proliferation” refers to division of cells orcell growth. Proliferation of CD4⁺,CD25⁺ T cells and CD4⁺,CD25⁻ T cellsmay be determined by any methods known in the art for measuringproliferation of lymphocyte populations. Examples of suitable methodsare described in, for example, Transplantation (1999) 67:605-613.

The specific antigen may be any antigen. For example, in the case oftesting for the development of tolerance to a transplant, the specificantigen will typically be a donor antigen. The donor antigen may bealloantigen or xenoantigen. In the case of testing for development oftolerance during autoimmune disease, the specific antigen will typicallybe the autoantigen. In the case of testing for tolerance to aninfectious agent, the specific antigen will typically be the infectiousagent or an antigen derived from that infectious agent. In the case oftesting for tolerance to a tumour, the specific agent will typically betumour antigen or tissue.

It will be appreciated by persons skilled in the art that the method maybe applied over a time period to determine whether tolerance to aspecific antigen is changing oyer time in the subject.

The inventors have further developed methods whereby CD4⁺,CD25⁺ T cellsactivated to a specific antigen, and therefore are capable of inducingtolerance to a specific antigen in a subject, can be cultured in vitro.This is made possible by the finding by the inventors that CD4⁺,CD25⁺ Tcells activated to the specific antigen can proliferate in the presenceof at least one cytokine selected from the group consisting of IL-5,IL-12, IL-23 and IFN-γ, or a biologically active fragment thereof, or afunctionally equivalent molecule thereof. Thus, the present inventionfurther provides a method of growing CD4⁺,CD25⁺ T cells activated to aspecific antigen in vitro by culturing the activated CD4⁺,CD25⁺ T cellsin the presence of at least one cytokine selected from the groupconsisting of IL-5, IL-12, IL-23 and IFN-γ, or a biologically activefragment thereof, or a functionally equivalent molecule thereof. Theactivated CD4⁺,CD25⁺ T cells are typically contacted with a specificantigen in vitro prior to, or simultaneously with, culturing theactivated CD4⁺,CD25⁺ T cells in the presence of the at least onecytokine selected from the group consisting of IL-5, IL-12, IL-23 andIFN-γ, or a biologically active fragment thereof, or a functionallyequivalent molecule thereof. The activated CD4⁺,CD25⁺ T cells may becultured in the presence of the specific antigen.

In some embodiments, the CD4⁺,CD25⁺ T cells activated to the specificantigen may be cultured in the presence of the at least one cytokineselected from the group consisting of IL-5, IL-12, IL-23 and IFN-γ, or abiologically active fragment thereof, or a functionally equivalentmolecule thereof, for a period of time longer than the time in whichactivated CD4⁺,CD25⁺ T cells remain viable in vitro following activationwhen cultured in the absence of at least one cytokine selected from thegroup consisting of IL-5, IL-12, IL-23 and IFN-γ, or a biologicallyactive fragment thereof, or a functionally equivalent molecule thereof.

The CD4⁺,CD25⁺ T cells activated to a specific antigen grown in vitromay be administered to a subject in need thereof to increase thesubject's tolerance to the specific antigen. The CD4⁺,CD25⁺ T cellsactivated to the specific antigen grown in vitro may be used to suppressthat portion of the immune system that recognises the specific antigen,but not other antigens. As the tolerance is increased to a specificantigen (rather than to antigens in general), that portion of the immunesystem that does not recognise the specific antigen is largelyunaffected. For example, tolerance may be induced to a specifictransplanted tissue, while at the same time the subject is capable ofeliciting an effective immune response to other antigens such asinfectious agents or transplanted tissue from an unrelated donor, etc.In other words, although the subject is tolerant to the specificantigens of the transplanted tissue, the subject is otherwise notimmunosuppressed. In contrast, known methods of ameliorating or reducingtransplant rejection in a subject involve immunosuppression of thesubject's immune system to all antigens, which renders the subjectsusceptible to infection from, for example, pathogens and opportunisticorganisms, and to cancer.

Thus, the invention further provides a method for increasing toleranceto a specific antigen in a subject which comprises administering to thesubject an effective amount of CD4⁺,CD25⁺ T cells activated to thespecific antigen wherein the activated CD4⁺,CD25⁺ T cells have beengrown in vitro by culturing activated CD4⁺,CD25⁺ T cells in the presenceof at least one cytokine selected from the group consisting of IL-5,IL-12, IL-23 and IFN-γ, or a biologically active fragment thereof, or afunctionally equivalent molecule thereof. As used herein, the expression“increasing tolerance to a specific antigen” means an increase intolerance to a specific antigen relative to the tolerance to thespecific antigen prior to application of the method of the invention.

The CD4⁺,CD25⁺ T cells activated to the specific antigen are typicallygrown in vitro by:

-   (a) contacting CD4⁺,CD25⁺ T cells activated to the specific antigen    with the specific antigen in vitro;-   (b) culturing the activated CD4⁺,CD25⁺ T cells in the presence of at    least one cytokine selected from the group consisting of IL-5,    IL-12, IL-23 and or a biologically active fragment thereof, or a    functionally equivalent molecule thereof.

The activated CD4⁺,CD25⁺ T cells may in some embodiments be cultured inthe presence of:

-   (a) at least one cytokine selected from the group consisting of    IL-5, IL-12 and IFN-γ, or a biologically active fragment thereof, or    a functionally equivalent molecule thereof; or-   (b) IL-5, a biologically active fragment thereof, or a functionally    equivalent molecules thereof; or-   (c) IL-12, a biologically active fragment thereof, or a functionally    equivalent molecules thereof; or-   (d) IFN-γ, a biologically active fragment thereof, or a functionally    equivalent molecules thereof; or-   (e) IL-23 a biologically active fragment thereof, or a functionally    equivalent molecules thereof.

The CD4⁺,CD25⁺ T cells activated to the specific antigen may beactivated in vitro or in vivo. In some embodiments, CD4⁺,CD25⁺ T cellsare isolated as naïve T cells and thereafter activated in vitro. Theterm “naïve CD4⁺,CD25⁺ T cell” refers to a CD4⁺,CD25⁺ T cell which hasnot been contacted by an antigen in the presence of IL-2 and/or IL-4,and is therefore not activated. Naïve CD4⁺,CD25⁺ T cells may be isolatedfrom thymus, bone marrow, peripheral lymphoid tissue or blood.Typically, the naïve CD4⁺,CD25⁺ T cells are isolated from the subject.Naïve CD4⁺,CD25⁺ T cells may be activated in vitro by contacting naïve Tcells with an antigen and culturing the T cells in the presence of oneor more of the cytokines selected from the group consisting of IL-2 andIL-4, or a biologically active fragment thereof, of a functionallyequivalent molecule thereof.

Thus, in one embodiment, the method of growing CD4⁺,CD25⁺ T cellsactivated to the specific antigen in vitro may comprise the followingsteps:

-   (a) contacting naïve CD4⁺,CD25⁺ T cells with a specific antigen and    culturing the lymphocytes in the presence of IL-2, a biologically    active fragment thereof, or a functionally equivalent molecule    thereof, and/or IL-4, a biologically active fragment thereof, or a    functionally equivalent molecule thereof, to thereby activate the    CD4⁺,CD25⁺ T cells; and-   (b) culturing the activated CD4⁺,CD25⁺ T cells in the presence of at    least one cytokine selected from the group consisting of IL-5,    IL-12, IL-23 and IFN-γ, or a biologically active fragment thereof,    or a functionally equivalent molecules thereof.

Steps (a) and (b) above may be performed simultaneously, or step (a) maybe performed prior to step (b).

Antigen presenting cells with the specific antigen presented on the cellsurface are typically co-cultured with the naïve CD4⁺,CD25⁺ T cells at37° C. in the presence of IL-2 and/or IL-4, biologically activefragments thereof, or functionally equivalent molecules thereof, for asufficient length of time to permit proliferation of CD4⁺,CD25⁺ T cellsactivated to the specific antigen.

In another embodiment, the CD4⁺,CD25⁺ T cells are activated in vivo andthereafter isolated for use in the method of the invention. In thisinstance, the CD4⁺,CD25⁺ T cells that are activated in vivo are isolatedfrom peripheral blood lymphocytes. Typically, the activated CD4⁺,CD25⁺ Tcells are isolated from the subject.

Without wishing to be bound by theory, the inventors believe that in thecase of CD4⁺,CD25⁺ T cells that are activated in vivo, following contactwith the antigen in vivo, activation of the resident naïve CD4⁺,CD25⁺ Tcells is thereafter supported by the presence of IL-2, a biologicallyactive fragment thereof, or a functionally equivalent molecule thereof,and/or IL-4, a biologically active fragment thereof, or a functionallyequivalent molecule thereof, in vivo. However, subsequent to thisactivation the activated CD4⁺,CD25⁺ T cells are less responsive tofurther contact with the antigen in the presence of IL-2, a biologicallyactive fragment thereof, or a functionally equivalent molecule thereof,and/or IL-4, a biologically active fragment thereof, or a functionallyequivalent molecule thereof.

In another embodiment, CD4⁺,CD25⁺ T cells activated to the specificantigen may be grown by:

-   -   (a) contacting a mixture of activated and naïve CD4⁺,CD25⁺ T        cells with the specific antigen in vitro;    -   (b) culturing the CD4⁺,CD25⁺T cells in the presence of IL-2, a        biologically active fragment thereof, or a functionally        equivalent molecule thereof, and/or IL-4, a biologically active        fragment thereof, or a functionally equivalent molecule thereof,        and at least one cytokine selected from the group consisting of        IL-5, IL-12, IL-23 and IFN-γ, or a biologically active fragment        thereof, or a functionally equivalent molecule thereof.

In this embodiment, the presence of IL-2 and/or IL-4, biologicallyactive fragments thereof, or functionally equivalent molecules thereof,permits activation of naïve CD4⁺,CD25⁺ T cells, while the presence ofthe at least one cytokine selected from the group consisting of IL-5,IL-12, IL-23 and IFN-γ, or a biologically active fragment thereof, or afunctionally equivalent molecule thereof, permits proliferation of theCD4⁺,CD25⁺ T cells activated to the specific antigen.

In some embodiments, the mixture of activated and naïve CD4⁺,CD25⁺ Tcells is contacted with the specific antigen in vitro and cultured inthe presence of IL-4, a biologically active fragment thereof, or afunctionally equivalent molecule thereof, IL-13, a biologically activefragment thereof, or a functionally equivalent molecule thereof, andIL-5, a biologically active fragment thereof, or a functionallyequivalent molecule thereof.

It is envisaged that it may not be immediately apparent whetherCD4⁺,CD25⁺ T cells from a subject are naïve or have been activated tothe specific antigen in vivo. Typically, a T cell may be determined tobe naïve or activated in vivo by contacting the T cell with the specificantigen, and determining proliferation of the T cell in the presence ofone or more cytokines selected from the group consisting of IL-5, IL-12,IL-23 and/or IFN-γ, or a biologically active fragment thereof, or afunctionally equivalent molecule thereof. Under such conditions, naïve Tcells will not undergo a proliferation while CD4⁺,CD25⁺ T cellsactivated to the specific antigen will exhibit proliferation.

Once the CD4⁺,CD25⁺ T cells are activated, whether it be in vitro or invivo, the CD4⁺,CD25⁺ T cells activated to the specific antigen are thencontacted with the specific antigen in a similar manner to thatdescribed above. That is, the CD4⁺,CD25⁺ T cells activated to thespecific antigen are contacted with the specific antigen by presentingthe antigen to the T cell on the surface of an antigen presenting cell.Typically, the antigen is presented to the T cell associated with amajor histocompatability (MHC) molecule (typically class II) on thesurface of an antigen presenting cell.

Subsequent to, or simultaneously with, contacting the specific antigen,the CD4⁺,CD25⁺ T cells are cultured in the presence of one or morecytokines selected from the group consisting of IL-5, IL-12, IL-23 andIFN-γ, or a biologically active fragment thereof, or a functionallyequivalent molecule thereof. As discussed above, activated CD4⁺,CD25⁺ Tcells are short-lived and normally do not remain viable for more than 3days. By culturing the activated CD4⁺,CD25⁺ T cells in the presence ofat least one cytokine selected from the group consisting of IL-5, IL-12,IL-23 and IFN-γ, or a biologically active fragments thereof, or afunctionally equivalent molecules thereof, the CD4⁺,CD25⁺ T cellsactivated to the specific antigen can undergo proliferation following orduring contact with the specific antigen. The step of culturing may beaccomplished by simply incubating the T cells in media containing thecytokines selected from the group consisting of IL-5, IL-12, IL-23 andIFN-γ, or a biologically active fragment thereof, or a functionallyequivalent molecule thereof, and typically, the antigen presenting cellwith specific antigen. For example, by culturing in the presence of acytokine selected from the group consisting of IL-5, IL-12, IL-23 andIFN-γ, or a biologically active fragment thereof, or a functionallyequivalent molecule thereof, the T cells are contacted with the cytokineselected from the group consisting of IL-5, IL-12, IL-23 and IFN-γ, or abiologically active fragment thereof, or a functionally equivalentmolecule thereof.

As used herein, the term “culturing” refers to the growth andmaintenance in a viable state of cells in vitro. The step of culturingCD4⁺,CD25⁺ T cells may be accomplished by simply incubating the T cellsin a culture medium which provides sufficient carbon, nitrogen, oxygenand other nutrients, growth factors, buffers, co-factors and any othersubstance as required to at least maintain the viability of the T cells.For example, T cells may be cultured in RPMI or DMEM supplemented with10% fetal calf-serum (FCS) and other supplements such as antimicrobialagents, growth factors, other cytokines (see, for example,Transplantation (1993) 55:374-379). Examples of suitable medium includemedium formulations that are known to those skilled in the art such as,for example, RPMI, IMDM, DMEM, DMEM/F12, EMEM with or without serum orwith reduced serum, and further optionally including antibiotics,lipids, transferrin, insulin, additional nutrient supplements such asamino acids and co-factors as required.

Generally, cultured T cells are incubated at 37° C. in a 5% CO₂atmosphere.

The CD4⁺,CD25⁺ T cells activated to a specific antigen are cultured inthe presence of one or more cytokines selected from the group consistingof IL-5, IL-12, IL-23 and IFN-γ, or a biologically active fragmentthereof, or a functionally equivalent molecule thereof. Typically, thecytokine selected from the group consisting of IL-5, IL-12, IL-23 andIFN-γ, or a biologically active fragment thereof, or a functionallyequivalent molecule thereof, is an isolated polypeptide that is addedexogenously to the media in which the T cells are cultured, either aspart of a culturing medium or as a purified polypeptide. Alternatively,the cytokine selected from the group consisting of IL-5, IL-12, IL-23and IFN-γ, or a biologically active fragment thereof, or a functionallyequivalent molecule thereof, may be the product of heterologous geneexpression in cells that are co-cultured with the T cells.

As discussed above, contacting the CD4⁺,CD25⁺ T cell activated to thespecific antigen with the specific antigen and culturing the T cells inthe presence of one or more cytokines selected from the group consistingof IL-5, IL-12, IL-23 and IFN-γ, or a biologically active fragmentthereof, or a functionally equivalent molecule thereof, results inproliferation of the CD4⁺,CD25⁺ T cells activated to the specificantigen.

In another embodiment, the CD4⁺,CD25⁺ T cells activated to the specificantigen may be contacted with the specific antigen as a portion of amixed population of cell lineages or as a portion of a mixed population,and the CD4⁺,CD25⁺ T cells isolated using the isolation methodsdescribed above.

Upon culturing in the presence of one or more cytokines selected fromthe group consisting of IL-5, IL-12, IL-23 and IFN-γ, or a biologicallyactive fragment thereof, or a functionally equivalent molecule thereof,and subsequent to proliferation, the T cells may then be administered tothe subject.

The T cells are administered to the subject to increase the number ofCD4⁺,CD25⁺ T cells activated to the specific antigen. The T cells aretypically administered by parenteral administration. Preparations forparenteral administration include suspensions in sterile aqueouscarriers. Aqueous carriers for suspensions may include saline andbuffered media. Parenteral vehicles include any solution which iscapable of maintaining the activity and viability of the lymphocytes,and may include, for example, cell culture medium, sodium chloridesolution, Ringer's dextrose, dextrose and sodium chloride, lactatedRinger's intravenous vehicles include fluid and nutrient replenishers,electrolyte replenishers (such as those based on Ringer's dextrose), andthe like. Preservatives and other additives may also be present such as,for example, anti-microbials, anti-oxidants, chelating agents, growthfactors and inert gases and the like.

The T cells can be administered, parenterally by injection or by gradualinfusion over time independently or together. Administration may beintravenously, intra-arterial, intraperitoneally, intramuscularly,intracavity, intraarticularly, or transdermally. Typically,administration is intravenously.

The administration may be local administration or regionaladministration to a site of immune activity.

Typically, the CD4⁺,CD25⁺ T cells activated to the specific antigen areadministered to obtain a ratio of CD4⁺,CD25⁺ T cells: CD4⁺,CD25⁻ T cellsin the subject of about 1:20. Typically, 1:10.

In another aspect, the invention provides a method for treating orpreventing in a subject in need thereof a disease resulting from animmune response to an antigen. For example, the disease may be anautoimmune disease, or host-versus-graft disease resulting fromallograft or xenograft rejection, or an allergic reaction.

In one embodiment, the disease is an autoimmune disease. As used herein,“autoimmune disease” refers to a disease resulting from an immuneresponse to an autoantigen. Autoimmune disease may include, but is notintended to be limited to, these particular types of autoimmunediseases: type 1 insulin dependent diabetes mellitis, inflammatory bowelsyndrome including ulcerative colitis and Crohn's disease, thromboticthrombocytopenic purpura, Sjogren's syndrome, encephalitis, acuteencaphalomyelitis, Guillain Barre Syndrome, chronic inflammatorydemyelination polyneuropathy, idiopathic pulmonary fibrosis/alveolitis,asthma, uveitis, iritis, optic neuritis, rheumatic fever, Reiter'ssyndrome, psoriasis, psoriasis arthritis, multiple sclerosis,progressive systemic sclerosis, primary biliary cirrhosis, pemphigus,pemphigoid, necrotising vasculitis, myasthenia gravis, polymyositis,sarcoidosis, granulomatosis, vasculitis, pernicious anemia, CNSinflammatory disorder, autoimmune haemolytic anaemia, Hashitomo'sthyroiditis, Graves disease, habitual spontaneous abortions, Raynaud'ssyndrome, dermatomyositis, chronic active hepatitis, celiac disease,autoimmune complications of AIDS, atrophic gastritis, ankylosingspondylitis, Addison's disease, atopic dermatitis, allergic rhinitis andconjunctivitis, asthma, chronic demyelinating neuropathy,glomerulonephritis including membranous nephropathy, focal sclerosingglomerulonephritis and minimal change nephropathy, systemic lupuserythematosis, scleroderma, rheumatoid arthritis, and juvenilearthritis.

Without wishing to be bound by theory, the inventors believe thatautoimmune disease may be the result of an imbalance in the ratio ofCD4⁺,CD25⁺ T cells to CD4⁺,CD25⁻ T cells. The present invention permitsthe ratio between these cells to be manipulated by culturing CD4⁺,CD25⁺T cells capable of inducing immune tolerance in vitro and subsequentlyintroducing these cells into the subject to increase the ratio ofCD4⁺,CD25⁺ T cells to CD4⁺,CD25⁻ T cells.

The ratio of CD4⁺,CD25⁺ T cells to CD4⁺,CD25⁻ T cells may be furtherincreased by administering to the subject an effective amount of atleast one cytokine selected from the group consisting of IL-5, IL-12,IL-23 and IFN-γ, or a biologically active fragment thereof, or afunctionally equivalent molecule thereof.

In another embodiment, the disease is a host-versus-graft diseaseresulting from allograft rejection. The term “allograft rejection” willbe understood by those skilled in the art as referring to an immuneresponse to an antigen(s) of a graft or transplanted tissue in a subjectwherein the graft or tissue is obtained from a different member of thesame species as the subject.

Allograft rejection includes rejection of all types of allograft and mayinclude for example, allografts of cornea, heart, valves, lung, kidney,liver, pancreas, pancreatic islets, brain, bone, intestine, skin, bonemarrow, stem cells, hematopoietic cell or other cells.

In yet another embodiment, the disease is a graft-versus-host diseaseresulting from bone marrow transplantation or other transplants orlympho-haemopoietic cells such as small bowel transplants.

In yet another embodiment, the disease is a host versus graft responseto a xenograft. The term “xenograft rejection” will be understood bythose skilled in the art as referring to an immune response to anantigen(s) of a graft or tissue transplant in a subject wherein thetissue is obtained from a member of a different species from thesubject.

Xenograft rejection includes rejection of all types of xenograft and mayinclude for example, xenografts of cornea, heart, lung, kidney, liver,pancreas, pancreatic islets, brain, bone, intestine, skin, valves, bonemarrow, stem cells, hematopoietic cells or other cells from, forexample, rodent, non-human primate, human, cattle, pig, sheep, camel,goat, kangaroo or horse.

In a further embodiment, the disease is an allergy. The term “allergy”will be understood by those skilled in the art to refer to a type Ihypersensitivity that is associated with a T cell response, typically aTh2 response, following contact with an allergen. The allergy may be anallergy to any allergen and includes, for example, asthma, eczema,atopic dermatitis, anaphylaxis, hayfever, allergic conjunctivitis,contact dermatitis, food allergy, drug or any other chemical allergy,fungal allergy, or an allergy as defined above to any other allergens orparts thereof.

Generally, the terms “treat”, “treating”, “treatment” and the like areused herein to mean affecting a subject, tissue or cell to obtain adesired pharmacologic and/or physiologic effect. The effect may beprophylactic in terms of completely or partially and/or may betherapeutic in preventing a disease or sign or symptom of disease termsof a partial or complete cure of a disease.

The disease may be treated by administering to the subject atherapeutically effective amount of a composition comprising theactivated CD4⁺,CD25⁺ T cells that have been grown in vitro according tothe methods described above and a pharmaceutically acceptable carrier.The composition comprising activated CD4⁺,CD25⁺ T cells that have beengrown in vitro according to the methods described above, may beadministered parenterally in formulations containing conventionalnon-toxic pharmaceutically acceptable carriers, adjuvants, and vehicles.

As used herein, the term “therapeutically effective amount” is meant asan amount effective to yield a desired therapeutic response. Forexample, an amount sufficient to prevent or treat autoimmune diseasesuch as those mentioned above.

The specific therapeutically effective amount will, obviously, vary withsuch factors as the particular condition being treated, the physicalcondition of the subject, the type of mammal being treated, the durationof the treatment, the nature of concurrent therapy (if any), and thespecific formulations employed and the relative constituent cellpopulations of the subjects immune system.

As used herein, a “pharmaceutically acceptable carrier” is apharmaceutically acceptable suspending agent, medium or vehicle fordelivering a therapeutic composition to a subject. Pharmaceuticallyacceptable carriers include aqueous solutions, non-toxic excipients,including salts, preservatives, buffers and the like, as described, forinstance, in Remington's Pharmaceutical Sciences, 15th ed. Easton: MackPublishing Co., 1405-1412, 1461-1487 (1975) and The National FormularyXIV., 14th ed. Washington: American Pharmaceutical Association (1975).The pH and exact concentration of the various components of thepharmaceutical composition are adjusted to maintain cell viability andactivity according to routine skills in the art. See Goodman andGilman's The Pharmacological Basis for Therapeutics (7th ed.).

Compositions comprising cytokines or antibodies can be administered, forin vivo application, parentally by injection or by gradual perfusionover time independently or together. Administration may beintravenously, intra-arterial, intraperitoneally, intramuscularly,intracavity, intraarticular, transdermally or subcutaneously. Typically,cytokines and antibodies are administered subcutaneously.

The compositions are preferably prepared and administered in dose units.For treatment of a subject, depending on activity of the compound,manner of administration, nature and severity of the disorder, age andbody weight of the subject, different daily doses can be used. Undercertain circumstances, however, higher or lower daily doses may beappropriate. The administration of the daily dose can be carried outboth by single administration in the form of an individual dose unit orelse several smaller dose units and also by multiple administration ofsubdivided doses at specific intervals.

The compositions according to the invention may be administeredsystemically, locally or regionally to a site of immune activity, in atherapeutically effective dose. Amounts effective for this use will, ofcourse, depend on the severity of the side effects and the weight andgeneral state of the subject. Typically, dosages used in vitro mayprovide useful guidance in the amounts useful for administration of thecomposition, and animal models may be used to determine effectivedosages for treatment of the cytotoxic side effects. Variousconsiderations are described, eg., in Langer, Science, 249: 1527,(1990).

The compositions may be in the form of a sterile injectable suspension.This suspension may be formulated according to known methods using thoseagents suitable for suspending and administering cell suspensions whichhave been mentioned above. Among the acceptable vehicles and solventsthat may be employed to suspend cells are cell culture medium, Ringer'ssolution, and isotonic sodium chloride solution.

Dosage levels of the composition of the present invention are of theorder of about 5×10⁶ to about 5×10⁹ cells per kilograM body weight, witha typical dosage range between about 5×10⁶ to about 5×10⁸ cells perkilogram body weight per day (from about 3×10⁸ cells to about 3×10¹¹cells per patient per day). The amount of cells that may be combinedwith the carrier materials to produce a single dosage will varydepending upon the host treated and the particular mode ofadministration. For example, a formulation intended for administrationto humans may contain about 5×10⁸ to 5×10¹⁰ cells with an appropriateand convenient amount of carrier material which may vary from about 5 to95 percent of the total composition. Dosage unit forms will generallycontain between from about 5×10⁸ to 10⁹ cells.

The inventors believe that the administration of naïve CD4⁺,CD25⁺ Tcells will suppress the activity of CD4⁺,CD25⁻ T cell, resulting in ageneral immune suppression to all antigens. The inventors believe thattolerance to a specific antigen may be increased using significantlyless CD4⁺,CD25⁺ T cells activated to the specific antigen than theamount of naïve CD4⁺,CD25⁺ T cells that would need to be administered toprovide a similar reduced immune response to the specific antigen.

It will be understood, however, that the specific dose level for anyparticular patient will depend upon a variety of factors including theactivity of the cells, the age, body weight, general health, sex, diet,time of administration, drug combination and the severity of theparticular disease undergoing therapy.

There are also circumstances in which tolerance to a specific antigen isundesirable. For example, tolerance to tumour cells, or to infectiousagents in chronic infections, is undesirable. The invention envisagesthat the requirement of CD4⁺,CD25⁺ T cells activated to the specificantigen for the presence of cytokines selected from the group consistingof IL-5, IL-12, IL-23 and IFN-γ, or a biologically active fragmentthereof, or a functionally equivalent molecule thereof, can be exploitedto reduce or break tolerance.

Thus, in a further aspect, the invention provides a method of reducingtolerance to a specific antigen which comprises depleting CD4⁺,CD25⁺ Tcells activated to a specific antigen in a subject by reducing oreliminating the activity of one or more cytokines selected from thegroup consisting of IL-5, IL-12, IL-23 and IFN-γ. The activity of IL-5,IL-12, IL-23 or IFN-γ may be reduced by any methods known in the art forreducing the activity of these cytokines in a subject. Typically, theactivity of IL-5, IL-12, IL-23 or IFN-γ may be reduced by administeringan effective amount of an antagonist of IL-5, IL-12, IL-23 and/or IFN-γ.Examples of antagonists include antibodies, soluble receptors orportions thereof, iRNA or siRNA, antisense molecules, etc. For example,the activity of IL-5, IL-12, IL-23 or IFN-γ may be reduced byadministering antibodies to IL-5, IL-12, IL-23 or IFN-γ. The tolerancemay be further reduced by administering an effective amount of IL-2and/or IL-4, biologically active fragments thereof, or functionallyequivalent molecules thereof. Without wishing to be bound by theory, theinventors believe that the administration of IL-2 and/or IL-4,biologically active fragments thereof, or functionally equivalentmolecules thereof, will activate naïve CD4⁺,CD25⁺ T cells, therebycommitting these T cells to a requirement for at least one cytokineselected from the group consisting of IL-5, IL-12, IL-23 and IFN-γ, or abiologically active fragment thereof, or a functionally equivalentmolecule thereof. Thus, in the absence of IL-5, IL-12, IL-23 and/orIFN-γ, biologically active fragments thereof, or functionally equivalentmolecules thereof, it is anticipated that activated CD4⁺,CD25⁺ T cellswill die, further reducing tolerance to the specific antigen.

Compositions comprising one or more of IL-2, IL-4, biologically activefragments thereof, or functionally equivalent molecules thereof, and/orantagonists of IL-5, IL-12, IL-23 and/or IFN-γ, such as antibodies, maybe prepared and administered as discussed above in relation tocompositions comprising CD4⁺,CD25⁺ T cells.

Also contemplated for use with the method of the invention are kits. Asused herein, the term “kit” refers to a group of components that arecapable of being used together in the methods of the invention. Forexample, the kit may be used to detect the presence of CD4⁺,CD25⁺ Tcells in a subject capable of inducing tolerance in the subject, or inother words, a diagnostic kit, or to prepare cells for inducingtolerance to an antigen in accordance with the invention, or toadminister antibody and/or cytokines to a subject. A kit may include,for example, one or more cytokines selected from the group consisting ofIL-5, IL-12, IL-23 and IFN-γ, or a biologically active fragment thereof,isolated protein or a medium such as, for example, a medium suitable forthe culturing of lymphocytes. The kit may further comprise one or morecytokines selected from the group consisting of IL-2 and IL-4. The kitmay further comprise antigen and or antigen presenting cells. The kitmay further include instructions for applying the method of theinvention using the components of the kit.

The kit may further comprise one or more specific antigens, such as, forexample, a plurality of antigens for testing of tolerance to variousallergens. The antigens of the kit may be present on the surface ofantigen presenting cells as described herein.

In another form, kit may include antibodies to one or more of thefollowing: IL-5, IL-12, IL-23 or IFN-γ, or a biologically activefragment thereof.

The kit may further comprise IL-2, a biologically active fragmentthereof, or a functionally equivalent molecule thereof, and/or IL-4, abiologically active fragment thereof, or a functionally equivalentmolecule thereof.

The invention will now be further described by way of reference only tothe following non-limiting examples. It should be understood, however,that the examples following are illustrative only, and should not betaken in any way as a restriction on the generality of the inventiondescribed above. In particular, while the invention is described indetail in relation to autoimmune disease, tissue rejection and allergicreactions, it will be clearly understood that the findings herein arenot limited to treatment of autoimmune disease, tissue rejection andallergic reactions.

EXAMPLES Example 1

These studies compared proliferation of CD4⁺ T cells to CD4⁺,CD25⁺ Tcells or CD4⁺,CD25⁻ T cell subsets or combinations of these CD4⁺ T cellsubsets in Mixed Lymphocyte Cultures (MLC). Cells were prepared fromlymph nodes and spleen of either naïve DA rats or DA rats that aretolerant to a fully allogenic PVG heterotopic heart graft. Tolerance wasdefined as non-rejection of the graft after 75 days in the absence ofmaintenance immunosuppression. Responses to self (DA), and to fully MHCincompatible specific donor (PVG) and fully MHC incompatible third party(Lewis) were compared. DA, PVG, Lewis share no MHC antigens and arefully allogeneic to each other. The nature of this tolerance haspreviously been described and can be induced in DA rats with PVGheterotopic cardiac allografts by a variety of therapies in the first 2weeks post transplant including cyclosporine, as well as either anti-CD4or anti-CD3 monoclonal antibody therapy (J. Exp Med. 1990, 171: 141,Transplantation, 1993, 55: 459, Transplantation 1997, 64: 1559). The MLCproliferative response of unfractionated peripheral lymphocytes tospecific donor stimulation is generally comparable to that to thirdparty (Transplantation, 1993, 55, 380-385).

Materials and Methods

Animals and procedures. DA (RT1^(a)), PVG (RT1^(c)), Lewis (RT1^(l)) andSprague Dawley rats were bred and maintained as previously described (J.Immunol. 1998, 161; 5146).

Operative procedures including heterotopic heart grafts, irradiation andpreparation of lymph node and spleen cells have been previouslydescribed (J. Exp. Med 1978, 148; 878). Heterotopic heart graft survivalwas monitored daily by palpation for loss of contraction and swellingassociated with rejection. Rejection was defined as total loss ofcontraction equivalent to that when there is total loss ofelectrocardiographic activity. Some grafts destined to become toleranthave transient swelling and reduced contractility, but recover. This isconsistent with an acute inflammatory response that may be required toinduce the specific tolerance mediating CD4⁺,CD25⁺ T cells.

Preparation of Peripheral Lymphoid Cells.

Single cell suspensions from spleen and lymph node cells (LNC) wereprepared, as described (34) and RBC lysed by a buffer of 0.83% NH₄Cl,0.1% KHCO₃ and 10 mM EDTA at pH 7.2. Cells were resuspended in PBS/2%BSA (MultiGel, Biosciences, Castle Hill, NSW, Australia).

Subsets were identified by mAb and indirect immunofluorescence staining,and analysis' on a FACScan, as described(35). Monoclonal antibodies usedwere R7.2 (TCR-α,β), G4.18 (CD3), W3/25 (CD4), MRCOx8 (CD8), MRCOx39(CD25, IL-2R alpha chain), L316 (CD122 IL-2R beta chain),(Pharmingen/Becton Dickenson, San Diego, Calif.).

Subsets of T cells were enriched by a combination of an indirect panningtechnique to deplete CD8⁺ T cells and B cells, as described (9) andMagnetic bead separation techniques and a MACS column, as described bythe manufacturer, (Miltenyi, Bergisch Gadenbach, Germany). Briefly,cells were incubated at 4° C. with optimised concentrations of MRCOx8(anti-CD8 monoclonal antibody) and MRC Ox33 (anti-CD45RA monoclonal thatbinds to B cells and other leucocytes except it does not bind T cells),washed three times with PBS/2% BSA then resuspended at 2×10⁷ cells/ml.These cells were incubated for 30 min at 4° C. on Petri dishes (GreinerLabortechnik, Frickenhausen, Germany) coated with rabbit anti-rat Ig andanti-mouse Ig (DAKO A/s. Glostrup, Denmark). This supernatant wasconcentrated in 85 μl of PBS and incubated for 4° C. for 15 minutes with13 μl of goat anti-mouse Ig micro-beads (Miltenyi) per 10⁶ cells. Afterwashing, cells were eluted on a CS MACS column (Milentyi) to obtain97-99% enrichment for CD4⁺ T cells. The enriched CD4⁺ T cells were thenincubated at 4° C. for 20 min with PE conjugated MRCOx39 (anti-CD25monoclonal antibody), then washed twice before incubation for 15 min at4° C. with 8 μl/10 ⁶ cells of mouse anti-PE mAb microbeads (Miltenyi).Cells were then eluted through a LS MACS column (Miltenyi) and wereeither resuspended in media with 20% Lewis rat serum for use in MLC orin PBS/2% BSA for injection to rats. The cells were 96-99% CD4⁺ and thedepleted population had <1% CD4⁺, CD25^(+high) T cells. The enrichedpopulation was 85-95% CD4⁺,CD25^(+high) T cells. For those experiencedin the art, it is known that enriched CD4⁺,CD25⁺ T cells populationsrefers to the CD4⁺,CD25^(+high) T cells, as separation techniquespreferentially enrich this population of CD4⁺,CD25⁺ T cells.

In some experiments CD4⁺,CD25⁺ T cells were directly enriched byincubation of unfractionated lymphoid cells at 4° C. for 20 min with PEconjugated MRCOx39, then washed twice before incubation for 15 min at 4°C. with mouse anti-PE mAb microbeads (Miltenyi). Cells were then elutedthrough a LS MACS column (Miltenyi) and were either resuspended in mediawith 20% Lewis rat serum for use in MLC or in PBS/2% BSA for injectionto rats.

Mixed Lymphocyte Cultures (MLC)

Stimulator cells were from thymus of rats given 8.5 gray whole bodyirradiation 24 hours before. This population of stimulator cells isdepleted of mature lymphocytes and is enriched for antigen presentingcells. Enriched antigen presenting cells are preferred as functionallymphoid cells will be stimulated and may produce cytokines that willactivate responder cells or may produce background stimulation.Stimulator cells from whole body′ irradiated donors have the peripheraland thymic lymphoid cells destroyed in vivo within 24 hours. Analternate method that could be used to enrich dendritic cells could bewith monoclonal antibody selection and Magnetic bead separation. Thestimulator cells can also be irradiated and left over night to allowperipheral lymphocytes to die of the effects of irradiation, leaving anantigen presenting cell enriched population. 10⁴ of these stimulatorcells were as effective as 2×10⁵ in vitro irradiated spleen cells. Thenormal ratio of responder to stimulator cells is 1:1 to 2:1 whenperipheral lymphoid cells are used as stimulators but when there isenrichment of antigen presenting cells by depletion of T and Blymphocytes then responders to stimulators cells may be 10-100:1.

Microcultures in U-bottom microtiter plates (Linbro, Flow Labs, VA) had2×10⁴ stimulators cells and either 2×10⁵ or 1×10⁵ responder cells/wellin a total volume of 200 μl. Usually there are 4-6 replicate wells setup for each experimental sample. Cell culture medium used was RPMI 1640(GIBCO, Grand Island, N.Y.) supplemented with 100 ng/ml penicillin, 100U/ml streptomycin (Glaxo, Boronia, Victoria, Australia), 2 mML-glutamine, 5×10⁻⁵M 2-mercaptoethanol (Sigma Chemicals, St. Louis,Mo.), and 20% Lewis rat serum. 20% Lewis rat serum produced lowbackground stimulation. Autologous or same species serum results in avery low background stimulation. This low background is due toelimination of the response to heterologous proteins in products such asfetal calf serum that are not used in the media.

In other experiments, serial dilutions of T cells subsets were culturedwith 2×10⁵ stimulators cells. Different ratios of mixtures of CD4⁺ Tcell subsets were also cultured with 2×10⁴ stimulators cells in limitingdilution assays.

Cells were cultured at 37° C. in humidified air containing 5% CO₂ and atvarious time points, usually at 3, 4, 5 and 6 days the cultures werepulsed with 0.5 μCi ³H-TdR (Amersham, Arlington Heights, Ill.) 16 hrprior to harvesting with a Titretek Cell Harvester (Flow Lab, Ayrshire,Scotland). Proliferation was assayed by adding liquid scintillationfluid before counting on a beta counter (1450 Microbeta Plus, BeckmanInstruments, Palo Alto, Calif.). Other wells were harvested at 24 and 48hours to extract mRNA for RT-PCR analysis of cytokine mRNA induction.

Results FIG. 1A

The results of measurement of the proliferation of naïve unfractionatedlymphocytes, and the enriched populations of CD4⁺,CD25⁺ T cells, CD4⁺ Tcells or CD4⁺,CD25⁻ T cells are shown in FIG. 1A. As can be seen fromFIG. 1A, the response at 4 and 5 days of CD4⁺ T cells is less that thatafter depletion of CD4⁺,CD25⁺ T cells as seen with the enrichedCD4⁺,CD25⁻ T cells. The response of CD4⁺,CD25⁺ T cells is much smallerthan with either unfractionated CD4⁺ T cells or CD4⁺,CD25⁻ T cells. Thiskinetic study demonstrates that the proliferation at day 4 and 5 is mostuseful, to demonstrate proliferative responses for unfractionated CD4⁺ Tcells and CD4⁺, CD25⁻ T cells. The response of naïve CD4⁺,CD25⁺ T cellspeaks earlier at day 3 and 4 and wanes by day 5 and 6, where as theresponse of unfractionated CD4⁺ T cells and of naïve CD4⁺,CD25⁻ T cellsdoes not appear until day 4 and peaks at days 5 or 6 before waning.

FIG. 1B

The results of comparison of proliferation to self, and two allogeneicstimulator strains of naïve CD4⁺ T cells, naïve CD4⁺,CD25⁺ T cells andnaïve CD4⁺,CD25⁻ T cells are shown in FIG. 1B. As can be seen from FIG.1B, the proliferative response at day 4 to syngeneic DA stimulators islow in all cases. The responses to PVG and Lewis are similar in eachsubtype of cells. As described above, the response of CD4⁺,CD25⁺ T cellsis less than the other populations and CD4⁺,CD25⁻ T cells have a greaterresponse than unfractionated CD4⁺ T cells.

FIG. 1C

FIG. 1C shows the result of serial dilution of naïve CD4⁺, CD4⁺,CD25⁺ orCD4⁺,CD25⁻ T cells. As can be seen from FIG. 1C, at all dilutions theresponse by CD4⁺,CD25⁻, T cells is greater that the equivalent number ofunfractionated CD4⁺ T cells. The unfractionated CD4⁺ T cells are a mixedpopulation with approximately 5% naïve CD4⁺,CD25⁺ T cells and 95%CD4⁺,CD25⁻ T cells. Thus the greater proliferation of CD4⁺,CD25⁻ T cellsis not simply due to an effect of enrichment (ie due to the loss of the5% CD4⁺,CD25⁺ T cells). It is consistent with an active effect of theminority population of naïve CD4⁺,CD25⁺ T cells inhibiting the majorpopulation of CD4⁺, CD25⁻ T cells.

FIG. 1D

The effect of admixing separated naïve CD4⁺,CD25⁺ T cells with separatednaïve CD4⁺,CD25⁻ T cells was examined and the result is shown in FIG.1D. As can be seen from FIG. 1D, naïve CD4⁺,CD25⁺ T cells had an activesuppressor effect CD4⁺,CD25⁺ T cells. In these experiments 1:10 mixes ofCD4⁺,CD25⁺ with CD4⁺, CD25⁻ T cells resulted in proliferative responsessimilar to the mixed population in CD4⁺ T cells where the ratio is1:10-1:20. Increasing the ratio to 1:1 resulted in near totalsuppression of the proliferative responses. This is consistent with thedemonstrated non-specific inhibitory effect of naïve CD4⁺,CD25⁺ T cellson immune responses in vitro.

FIG. 1E

Illustrates the proliferation of CD4⁺ T cells, CD4⁺,CD25⁺ T cells orCD4⁺,CD25⁻ T cells from tolerant animals. The response at 4 and 5 daysof CD4⁺ T cells is similar to the tolerated strain (PVG) as it is tothird party Lewis. After depletion of CD4⁺,CD25⁺ T cells, as seen withthe enriched CD4⁺,CD25⁻ T cells, the proliferative response to thetolerated strain is less than to the third party strain.

The response of CD4⁺,CD25⁺ T cells is much smaller than with eitherunfractionated CD4⁺ or CD4⁺,CD25⁻ T cells. These CD4⁺,CD25⁺ T cells donot respond to specific donor in that their response is similar to theresponse to self (DA). These CD4⁺,CD25⁺ T cells from tolerant animalsretain their response to third party, which is greater than to eitherspecific donor or self.

The response to self (DA) is less in all cultures than that to fullyallogeneic stimulators, PVG or Lewis. The response to PVG and Lewiswhich are MHC unrelated strains is similar for unfractionated CD4⁺ Tcells, but is less to PVG than to Lewis for both CD4⁺,CD25⁺ T cells andCD4⁺,CD25⁻ T cells.

These results suggested that the CD4⁺,CD25⁺ T cells from tolerantanimals die in standard culture conditions. Thus they do not inhibit theCD4⁺,CD25⁻ T cells in the mixed population of unfractionated CD4⁺ Tcells, thereby their removal does not lead to an enhanced response ofenriched CD4⁺,CD25⁻ T cells. Alone CD4⁺,CD25⁺ T cells do not respond todonor alloantigen, again consistent with them not surviving, and theirpossible dependence on cytokines for growth.

FIG. 1F

Shows serial dilution of CD4⁺, CD4⁺,CD25⁺ T cells or CD4⁺,CD25⁻ T cellsfrom DA rats tolerant to PVG heart allografts. At all dilutions theresponse to specific donor (PVG) by CD4⁺,CD25⁻ T cells is less that theresponse to third party (Lewis) and no greater than that to self (DA).In contrast, with unfractionated peripheral lymphocytes or with CD4⁺ Tcells the response to specific donor (PVG) is similar to third party(Lewis) and greater than to self (DA). At all dilutions the response ofCD4⁺,CD25⁻ T cells to third party (Lewis) or to self (DA) is muchgreater than the equivalent number of unfractionated CD4⁺ T cells.

This study demonstrates that the response to PVG by CD4⁺,CD25⁻ T cellscompared to Lewis is reduced and is different to that observed withnaïve CD4⁺,CD25⁻ T cells where there was a similar increase to both PVGand Lewis above that observed with unfractionated CD4⁺ T cells. Further,naïve CD4⁺,CD25⁻ T cells response to PVG is always greater than to self.

This study suggested that the response of unfractionated CD4⁺ T cellsfrom tolerant animals is due to a diminished response of the CD4⁺,CD25⁻Tcells to PVG being unchecked by CD4⁺, CD25⁺ T cells in the absence ofcytokines capable of stimulating activation of CD4⁺,CD25⁺ T cells or ofprolonging survival or stimulating proliferation of activated CD4⁺CD25⁺T cells. In contrast CD4⁺,CD25⁺ T cells from animals tolerant to PVGretain the capacity to inhibit the response to third party Lewis andtheir removal allowed an enhanced response of CD4⁺,CD25⁺ T cells toLewis compared to unfractionated CD4⁺ T cells.

These studies demonstrated that there is a difference in the CD4⁺,CD25⁺,T cells in tolerant animals compared to naïve. This is consistent withthe CD4⁺,CD25⁺ T cell from tolerant host dying in culture withoutcritical growth factors, and that it thereby cannot suppress in thisassay. Alternately the activation of CD4⁺,CD25⁻ T cells results in achange of cytokine milieu that destroys rather than promotes thefunction of specific CD4⁺,CD25⁺ T cells that are maintaining tolerance.That is, new activation and release of early T cell activation cytokinesdestroys the specific tolerance mediating CD4⁺,CD25⁺ T cells.

Conclusions

In mixed lymphocyte cultures (MLC), the proliferative response ofCD4⁺,CD25⁺ T cells:

-   -   a) to MHC incompatible alloantigens can be reproducibly assayed        with the defined culture conditions that eliminate any        background.    -   b) from naïve animals is only to MHC incompatible stimulators,        not to self.    -   c) from naïve animals inhibits the proliferation of naïve        CD4⁺,CD25⁻ T cells.    -   d) from tolerant animals is only to third party and not to        specific donor or self.    -   e) from tolerant animals cannot inhibit the response of tolerant        CD4⁺,CD25⁻ T cells to specific donor but can inhibit the        response to third party.    -   f) From naïve animals has a non-alloantigen specific suppressor        effect, in distinction from those from tolerant animals that        retain the capacity to suppress response to third party        alloantigen but not to specific donor alloantigens in the        absence of cytokines capable of stimulating activation of        CD4⁺,CD24⁺ T cells or of prolonging survival or stimulating        proliferation of activated CD4⁺CD25⁺ T cells.

Example 2

The following experiments examined the effects of various cytokines onthe proliferative response in MLC of CD4⁺,CD25⁺ T cells, and theresponse of naïve and tolerant enriched CD25⁺,CD4⁺ T cells to individualcytokines was compared.

Materials and Methods

Cytokines were produced as described, and include IL-2 (32, 33) and oneunit was defined as that required to induce 50% of maximal proliferationof the IL-2 dependent CTLL line. The cloning production and assaying ofthese cytokines has been described and used standard methods fortransfection into CHO-K1 cells (Transplantation Proc. (1999) 31,1574-5,1999 and 31, 1572, 1999) and included IL-4, IL-5, IFN-γ, IL-10, IL-12(p70), IL-12 (p40), IL-13. Human TGF-β was purchased from Sigma.Cytokine was added to each relevant well as a 50 μl aliquot of CHO-Ktransfected cell line supernatant. These supernatants have 5000-50,000units per ml. Thus the relevant cytokines were at a final concentrationof 1000-12,000 units per ml. Controls had supernatant from a nontransfected CHO-k1 cell line added to the medium.

Reverse Transcription—Polymerase Chain Reaction (RT-PCR):

The methods for mRNA extraction, cDNA synthesis and semi-quantitativePCR have been described (31, 35). All samples were standardized byquantitation of cDNA by spectroscopy and by PCR of the house-keepinggene GAPDH. The primers and optimal cycle conditions for the cytokinesIL-2, IL-4, IL-5, IL-10, IFN-γ, TNF-α, have been described(Transplantation 1998; 1145-1142). Primers for IFN-γ receptor andIL-5-receptor alpha chain were designed and validated. Reaction andamplification conditions were optimised for each primer set using a PCRmachine (Corbett Research, Sydney, NSW, Australia). PCR product was thenanalysed by electrophoresis on 6% polyacrylamide gels and stained withethidium bromide. The specificity of the RT-PCR products were verifiedby Southern transfer and hybridisation with dideoxygenin (DIG) 3′-endlabelled oligonucleotide probes. Hybridised probe was detected using theDIG luminescent detection kit (Boehringer Mannheim). The methods for thesemi-quantitative technique RT-PCR used were as previously described(Transplantation 1997, 64; 1559-1567). Starting cDNA samples wereassayed at neat, 10, 100 and 1000 fold dilutions and reactionsterminated at a cycle in the optimum range for each product. For mostsamples there were duplicate samples assayed at each dilution. Levels ofmRNA expression were compared using the lowest dilution at which the PCRproduct was detected. Negative controls without cDNA, and positivecontrols of cDNA from ConA stimulated rat lymphocytes, were included inall experiments.

Results

Enriched CD4⁺,CD25⁺ T cells were cultured in MLC against self (DA),specific donor (PVG) and third party (Lewis) stimulators. Cultures wereassayed for proliferation at 3-4 days as described above.

FIG. 2A

FIG. 2A illustrates the response of naïve DA cells to either DA antigenor PVG antigen in the presence of cytokines as indicated. As can be seenfrom FIG. 2A, only IL-2 and IL-4 resulted in enhanced proliferation ofnaïve CD4⁺,CD25⁺ T cells. These oytokines enhance proliferation to self,and both allogeneic stimulators, to a similar degree. This is consistentwith poly-clonal activation by either IL-2, or IL-4. Addition of IL-5,IL-10, IL-13, IFN-γ did not enhance proliferation of naïve CD4⁺,CD25⁺ Tcells. Note the background response to self is at approx 100 cpm,equivalent to counts obtained with distilled water. That is with themethods used, (as described in FIG. 1) there was no non-specificproliferation to extraneous factors like media, or stimulator cells. Thebackground counts are equivalent to having distilled water assayed.

FIG. 2B

FIG. 2B is a graph showing the proliferation at day 3 of activatedCD4⁺,CD25⁺ T cells against self-antigen (black) or donor antigen (grey)in the presence of TGF-β (D), IFN-γ (E), IL-12p70 (F), IL-5 (G), orIL-10 (H). FIG. 2B shows that the response of activated CD4⁺,CD25⁺ Tcells is different to that of naïve CD4⁺,CD25⁺ T cells in severalaspects. First as described in FIG. 1E, activated CD4⁺,CD25⁺ T cells donot proliferate to specific donor in the absence of specific cytokinesand thereby differ from naïve CD4⁺,CD25⁺ T cells. Activated CD4⁺,CD25⁺ Tcells proliferate to third party Lewis in a manner similar to naïveCD4⁺,CD25⁺ T cells. Both activated and naïve CD4⁺,CD25⁺ T cells do notproliferate to self (DA).

The addition of IL-2 or IL-4 to cultures resulted in polyclonalactivation, like that observed with naïve CD4⁺, CD25⁺ T cells (data notshown), in that there was a marked enhanced proliferative response ofnaïve CD4⁺,CD25⁺ T cells to self (DA), specific donor (PVG) and thirdparty (Lewis).

The response to other cytokines identified a different pattern foractivated CD4⁺,CD25⁺ T cells to the specific donor PVG for somecytokines but not others. Activated CD4⁺,CD25⁺ T cells response to bothself (DA) and third party Lewis stimulators were similar to that ofnaïve cells for all cytokines.

Thus these experiments demonstrated a different response to PVG (donoror specific antigen), that was most consistent with IFN-γ and IL-5.IL-12 (p70) and TGF-β also showed an effect. Control cultures and thosewith cytokines that did not induce proliferation, had responses atbackground, approx. 100 cpm.

These experiments demonstrated that CD4⁺,CD25⁺ T cells activated to aspecific antigen require either IL-5, IL-12 or IFN-γ to grow andsurvive.

Conclusions

Cytokines have different effects on naïve and tolerant CD4⁺,CD25⁺ Tcells.

-   -   a) IL-2 and IL-4 markedly enhance proliferation of CD4⁺,CD25⁺ T        cells from naïve or tolerant cell donors to self, specific donor        and third party.    -   b) IL-5 and IFN-γ only enhances proliferation of tolerant        CD4⁺,CD25⁺ T cells to specific donor and not to self or third        party. They have no effect on naïve CD4⁺,CD25⁺ T cells        proliferation to self or to alloantigens.

Example 3

These studies examine the ability of CD4⁺,CD25⁺ T cells to transfertolerance to a host.

Materials and Methods Adoptive Transfer Assays.

These were conducted as described (J. Exp. Med 1978, 148; 878-889 andTransplantation 1993, 55; 374-379). Briefly, DA rats were irradiatedwith 7.5-8.5 gray from a ⁶⁰Co source, then grafted with heterotopicadult heart from a PVG donor that had also been irradiated (FIG. 3 A).This source of irradiation required treatment over 15-30 minutes, asdescribed (J. Exp. Med 1978, 148; 878-889). In the later experiments theirradiation source was a linear accelerator and whole body irradiationwas delivered in 2-3 minutes. These irradiated hosts were given aheterotopic heart graft and also were restored within twenty-four hoursof irradiation with CD4⁺ T cells, either unfractionated or enriched forCD4⁺,CD25⁻ T cells or CD4⁺,CD25⁺ T cells. Heart graft rejection ismonitored initially by daily palpation and if there is doubt by ECGmonitoring. Rejection is scored when there is loss of all palpablecontraction that is equivalent to total loss of electrocardiographactivity.

Results

FIGS. 3A and 3B show the capacity of naïve CDe T cells to effectrejection response on adoptive transfer to irradiated hosts.

For the studies reported in FIG. 3B a semi-quantitative scale was usedwhere a score of 4+ indicated robust contraction with normal auxiliaryheart graft rate. 3+ indicated minor slowing and or reducedcontractility. 2+ indicated obvious slowing and swelling of the graftbut clear palpation of beat. 1+ indicated marked slowing and poorlypalpable beat, usually associated with markedly reduced amplitude of theECG. 0 indicated no palpable contraction and equivalent to no detectableECG activity.

FIG. 3A

FIG. 3A shows a comparison of rejection time in adoptive irradiated DArats restored with different doses of naïve CD4⁺ cells. Data combinedfrom all laboratories and sites of Prof B. Hall. Group A, n=7; Group B,n=17; Group C, n=86; Group D, n=6. To alter the balance of cells in ahost, adoptive transfer experiments were performed where the DA hostsown lymphocytes were destroyed by near lethal whole body irradiation(700-850 rads). In this model, heterotopic PVG heart grafts are notrejected by the whole body irradiated hosts, whilst in non irradiatedhosts rejection occurs in 6-9 days which is first set rejection time.Restoration of the irradiated host with an enriched population of CD4⁺ Tcells restores a rejection response, but not to the normal first settime. There is no dose response with these cells, in that 5 million CD4⁺T cells are as effective as 20 or 100 million CD4⁺ T cells. Therejection time on average is >12 days with these cells.

TABLE 1 Rejection of PVG heterotopic cardiac allografts in irradiated DArecipients restored with unfractionated CD4⁺ T cells and/orsubpopulations of CD4⁺ T cells Restorative Graft rejection signifi-Irradi- Inoculata times in days cance* ation Subtype Number medianactual p 1 − — — 8 7 (3), 8 (5), <0.001   9 (1) 2 + — — >100 >100 (10) <0.001 3 + CD4⁺ 5 × 10⁶ 11 8, 11(11), 13, 15, >100 4 + CD4⁺, CD25⁻ 5 ×10⁶ 9 8, 9 (3), <0.001  10 (3) 5 + CD4⁺, CD25⁺ 5 × 10⁶ >100 >100(5)<0.001 6 + CD4⁺CD25⁺, 5 × 10⁶ >100 >100(5) <0.001 CD4⁺ 5 × 10⁶ 7 +cultured 5 × 10⁶ >100 >100(3) <0.01 IL-2 5 × 10⁶ CD4⁺, CD25⁺ CD4⁺ 8 +cultured 5 × 10⁶ >100 >100(3) <0.01 IL-2 CD4⁺, CD25⁺ *Compared to 5 ×10⁶ CD4⁺ T cells (see row 3)

The effects of enriched naïve CD4⁺ T cells, CD4⁺,CD25⁺ T cells orCD4⁺,CD25⁻ T cells in the adoptive DA host with a PVG allograft werecompared and the results are shown in Table 1. As in FIG. 3A,unfractionated naïve CD4⁺ T cells did not restore first-set rejectiontime but restored rejection to a median of 11 days. In this model theirradiation source was different to that in FIG. 1. CD4⁺,CD25⁺ T cellshad no capacity to effect rejection, and no grafts rejected. CD4⁺,CD25⁻T cell restored rejection to near first-set tempo. Admixing naïveCD4⁺,CD25⁺ T cells with naïve CD4⁺ T cells in a ratio of 1:1 preventedrestoration of rejection, and the grafts appeared to function without asignificant rejection episode for over 100 days. CD4⁺,CD25⁺ T cellsexpanded by culture for three days with donor antigen (PVG) and IL-2 toexpand the numbers suppressed naïve CD4⁺ T cells. This was done asdescribed in methods for mixed lymphocyte culture, but in bulk in 50 mltissue culture flasks. These results demonstrate that naïve CD4⁺,CD25⁺ Tcells can be cultured with donor antigen and expanded in vitro tomaintain suppressor function and not acquire the capacity to effectrejection.

FIG. 3B

The effects of varying the ratios of naïve CD25⁺,CD4⁺ T cells to naïveCD25⁻,CD4⁺ T cells in the restorative inoculum on rejection times inirradiated adoptive hosts was examined and a graph of the results isshown in FIG. 3B.

FIG. 3B illustrates heart graft function up to 50 dayspost-transplantation in rats following administration of; A, 5×10⁶ naïveCD4⁺ T cells (closed triangles) (n=9); B, 20×10⁶ naïve CD4⁺ T cells(closed circles) (n=4); C, 5×10⁶ naïve CD4⁺,CD25⁻ T cells (opentriangles) (n=4); D, 0.5×10⁶ naïve CD4⁺,CD25⁺ T cells plus 5×10⁶ naïveCD4⁺ T cells (open squares) (n=9): E, 5×10⁶ naïve CD4⁺,CD25⁺ T cellsplus 5×10⁶ naïve CD4⁺ T cells (closed squares) (n=9). On this scale ratsgiven 20 million CD4⁺ T cells effected rejection most rapidly and thosegiven CD4⁺,CD25⁻ T cells had a similar rapidity of rejection. Thosegiven 5 million CD4⁺ T cells alone rejected less rapidly than thoserestored with CD25⁻, CD4⁺ T cells. Mixing 0.5 million CD4⁺,CD25⁺ T cellswith 5 million CD4⁺ T cells further slowed the rejection processes, inthat grafts were not totally rejected but had poor function long term.In these animals the ratio of CD25⁺, CD4⁺ T cells: CD25⁻, CD4⁺ T cellswas 1:5-10 compared to 1:20-40 in those given unfractionated cells.

Mixing 5 million CD4⁺,CD25⁺ T cells with 5 million CD4⁺ T cells totallysuppressed rejection with all grafts surviving indefinitely with verygood function.

These studies demonstrate that the naïve CD4⁺,CD25⁺ T cells can suppressnaïve CD4⁺, CD25⁻ T cells if present in a ratio approaching 1:1.

Further it shows that expansion of CD4⁺, CD25⁺ T cells by culture withIL-2 may allow production of sufficient self suppressor T cells toachieve such ratios, if the host is depleted of CD4⁺,CD25⁻ T cells orthe CD4⁺,CD25⁻ T cells function is impaired. Culture of naïve CD4⁺,CD25⁺T cells with donor antigen does not convert these cells into ones thatcan effect rejection. Thus culture of naïve CD4⁺,CD25⁺ T cells mayexpand the number of suppressor CD4⁺,CD25⁺ T cells. Treatment ofunfractionated CD4⁺ T cells in vitro with IL-4, does not enhance thetolerance effect of that CD4⁺,CD25⁺ T cells, indicating enrichedCD4⁺,CD25⁺ T cells must be used. (Data not shown)

Conclusions

Altering the balance of CD4⁺,CD25⁺ T cells to CD4⁺,CD25⁻ T cells in vivocan lead to tolerance, manifest by experiments with naïve cells used torestore rejection in irradiated hosts;

-   -   a) Naïve unfractionated CD4⁺ T cells do not show a dose response        effect, suggesting there is a homeostatic balance of the 1-10%        CD4⁺,CD25⁺ T cells against the >90% CD4⁺,CD25⁻ T cells.    -   b) Removal of the 1-10% CD4⁺,CD25⁺ T cells, allows the        CD4⁺,CD25⁻ T cells to effect rapid rejection, confirming that        the naïve CD4⁺,CD25⁺ T cells in their natural mixed population        in naïve CD4⁺ T cells function to moderate rejection.    -   c) CD4⁺,CD25⁺ T cells alone do not effect rejection and can        inhibit rejection when the ratio of naïve CD4⁺,CD25⁺ T cells to        naïve CD4⁺,CD25⁻ T cells is increased, with near total        suppression of rejection when the ratio is 1:1.    -   d) Naïve CD4⁺,CD25⁺ T cells when expanded in MLC with specific        donor antigen and IL-2 retain the capacity to suppress at 1:1        ratio and do not revert to cells that effect rejection.

Example 4

This study examines proliferation of CD4⁺,CD25⁺ T cells followingcontact with antigen in the presence of IL-2 or IL-4.

FIG. 4A

FIG. 4A shows the results of culture of naïve CD4⁺,CD25⁺ T cells from DArats with no stimulator cells ie. no antigen (first column), selfstimulator cells ie. antigen presenting cells presenting antigen from DArats (middle column), or allogeneic PVG stimulator cells ie. antigenpresenting cells presenting antigen from PVG rats.

The top panel of FIG. 4A shows the effect of contacting naïve CD4⁺,CD25⁺T cells with antigen in the presence of IL-2. As can be seen from FIG.2, contact with PVG antigen in the presence of IL-2 resulted inincreased proliferation in all assays, with greater effect at day 5 thanday 4. The proliferation to self and allogeneic stimulator cells, wassimilar, and that to no stimulators much less.

The bottom panel of FIG. 4A shows the effect of contacting naïveCD4⁺,CD25⁺ T cells with antigen in the presence of IL-4. The response toallogeneic was greater than that to self or those with no stimulators.This suggests the effect of culturing CD4⁺,CD25⁺ T cells in the presenceof IL-4 is greater when the cells are contacted with specific antigenrather than with self antigen.

FIG. 4B

FIG. 4B illustrates a time course of proliferation of naïve CD4⁺,CD25⁺ Tcells from DA rats cultured with allogeneic PVG stimulator cells. Cellscultured with no cytokine supplementation had peak proliferation at day3 as described in FIG. 1A. No proliferation was detectable at day 5 andday 6 with counts at background (equal to distilled water counts <150ccpm). IL-2 induced marked proliferation at all days with a peak at day5. IL-4 enhanced proliferation on all days and this proliferation peakedat day 4. Profiles of the phenotype of these cells after 3-4 days inculture shows there is an emergence of a double positive population thatexpresses both CD8 and CD4 (10-35% of cells) and continue to expressCD25. The original cells were <1% CD4⁺, CD8⁺ cells, and <2% CD8⁺ Tcells.

FIG. 4C

FIG. 4C shows the result of a semi-quantitative RT-PCR assay for mRNAexpression of cytokines and cytokine receptors by CD4⁺,CD25⁺ T cellscultured with IL-2 and IL-4.

Two experiments are shown in which naïve CD4⁺,CD25⁺ T cells werecultured in MLC with IL-2, IL-4 or controls with CHO-k1 cellsupernatants. Culture was for 3 days before mRNA was extracted.Semi-quantitative RT-PCR was performed as described (Transplantation,1997, 64, 1559-1567). The samples are serial dilutions of cDNA fromright to left. The results show that there was no induction of IL-2 byany treatment, but abundant mRNA for IL-4, IL-5, IL-10, IL-13, IFN-γ andinducible nitric oxide synthetase. Those cells cultured with IL-2expressed IL-12Rβ2 that was not expressed in those cultured with IL-4.With IL-2 there was no IL-5Rα expression but in other experiments thisreceptor is induced by culture with IL-4.

FIG. 4D

FIG. 4D illustrates cytokine and cytokine receptor induction in culturedtolerant CD4⁺,CD25⁺ T cells. This compares tolerant cells stimulated byspecific donor, third party stimulators or self stimulators. Culturescontained no cytokine supplement (CTL top three lines) or weresupplemented with IL-2, IL-4 or CHO-k supernatant. −&+CTLS were cDNAfrom ConA activated T cells was used as a control for the assay itself(−&+controls). mRNA was assayed using semi-quantitative RT-PCR withserial dilutions of cDNA from right to left.

There was no induction of IL-2 mRNA in any CD4⁺,CD25⁺ T cellspreparation. The most consistent and abundant mRNA was for IFN-γ thatwas detected in all assays and tended to be enhanced after culture ofCD4⁺,CD25⁺ T cells with either IL-2 or IL-4. IL-12β2 was induced inCD4⁺,CD25⁺ T cells cultured with IL-2 and to a lesser extent with IL-4,and was not observed in CD4⁺,CD25⁺ T cells cultured with no cytokines.IFN-γ mRNA was induced in cells cultured with IL-4.

mRNA for IL-4 and IL-5 was only mainly induced in CD4⁺,CD25⁺ T cellsexposed to IL-2 or IL-4 and in neither control culture with no cytokinesor CHO-k supernatant. IL-13 mRNA was detected in all samples with someinduction with IL-2 and IL-4, but this may in part reflect differencesin starting cDNA as per GAPDH levels. mRNA for IL-10 and TGF-β werepresent in all cultures and were not discriminatory. mRNA for IL-5Rα wasinduced in CD4⁺,CD25⁺ T cells exposed to IL-4 as indicated by very faintbands. Samples assayed at day 4 show a more definite expression ofIL-5Rα mRNA in cells cultured with IL-4 mRNA. See FIG. 4E.

This suggests a different population of T suppressor cells may beinduced with IL-2 and with IL-4, the IL-2 inducing cells that mightrespond to Th1 cytokines and IL-4 ones that might respond to Th2cytokines such as IL-5.

FIG. 4E.

FIG. 4E shows the IL-5-Rα chain is induced in CD4⁺,CD25⁺ T cellscultured with IL-4. In this study naïve CD4⁺,CD25⁺ T cells from DA ratswere cultured with either self DA stimulators or PVG stimulators for 4days. IL-5Rα mRNA was assayed in cDNA collected from cells. Cells werecultured with nil cytokine, IL-2 or IL-4. IL-5Rα mRNA was only detectedin cells cultured with IL-4 with greater levels in those cultured withalloantigen than those cultured with self antigen. Controls wereuncultured CD4⁺,CD25⁺ T cells, cDNA from ConA activated lymphocytes anda known cDNA which had IL-5Rα.

FIG. 4F

FIG. 4F shows real time RT-PCR for IFN-γ receptor (IFNGR) on CD4⁺,CD25⁺T cells contacted with antigen in the presence of IL-2.

This study clearly demonstrated that there was induction of IFN-γreceptor on CD4⁺,CD25⁺ T cells cultured with IL-2 and alloantigeneic PVGstimulators and to a lesser degree with self DA stimulators, and not incells cultured with no cytokines.

Taken together, these studies in FIGS. 4A, 4B and 4C show thatCD4⁺,CD25⁺ T cells exposed and activated with Th1 responses (ie IL-2)and alloantigen, developed receptors that would allow them to respond toTh1 cytokines such as IFN-γ and IL-12. On the other hand, CD4⁺,CD25⁺ Tcells exposed and activated by Th2 cells (ie IL-4) in the presence ofalloantigen develop receptors for Th2 cytokines such as IL-5. They alsoexpress the Th1 cytokine IFN-γ mRNA. These results are consistent withour observation that activated CD4⁺,CD25⁺ T cells from rats withtolerance to PVG proliferate to PVG but not self or third party wheneither IFN-γ or IL-5 are added to the culture, but not if there is nocytokines.

The inventors propose that under normal circumstances of an immuneresponse, with early activation there would be both IL-2 and IL-4resulting in activation of both types of CD4⁺,CD25⁺ T cells.

The inventors propose to call these two types of activated CD4⁺,CD25⁺ Tcells Ts1 cells and Ts2 cells. Ts1 cells are CD4⁺,CD25⁺ T cellsactivated by IL-2 that acquire responsiveness to IFN-γ and/or IL-12p70and Ts2 cells that are CD4⁺,CD25⁺ T cells activated by IL-4 that acquireresponsiveness to IL-5. Ts refers to suppressor T cells of theCD4⁺,CD25⁺ T cell phenotype.

Conclusions

IL-2 and IL-4 have different effects on CD4⁺,CD25⁺ T cells in culture,including;

-   -   a) Whilst both stimulate proliferation to self and alloantigen,        the response to IL-4 peaks and tapers earlier than that with        IL-2.    -   b) Culture with IL-2 leads to development of a CD4⁺,CD8⁺ double        positive CD25⁺ T cells. This development of double positive        cells is probably de-differentiation of CD4⁺,CD25⁺ T cells to        re-express CD8.    -   c) Neither culture with IL-2 or IL-4 induces detectable        expression of IL-2 mRNA, but both IL-2 and IL-4 induce mRNA for        IL-4, IL-5, IL-12p40, iNOS. There appears to be no detectable        specific induction of IL-10, IL-13 and TGF-β.    -   d) There is late expression of new cytokine receptors, with        IFN-γR appearing when cultured with IL-2 and antigen. IL-5R        appears with IL-4. IL-12R(32 appears with both stimuli but        possibly more with IL-2 than IL-4.    -   e) The pattern of cytokine mRNA expression with tolerant cells        cultured with IL-2 or IL-4 is similar to that with naïve        CD4⁺,CD25⁺ T cells, consistent with this being polyclonal        activation of CD4⁺,CD25⁺ T cells, not activation of alloantigen        specific CD4⁺,CD25⁺ T cells. f) That IL-2 induces an        IFN-γ/IL-12p70 dependent suppressor cell, that we propose to        ascribe the name Ts1.    -   g) That IL-4 induces an IL-5 dependent suppressor cell, that we        wish to ascribe the name Ts2.

Example 5

This study examines the effect of naïve CD4⁺,CD25⁺ T cells on CD4⁺,CD25⁻T cells.

FIG. 5A

FIG. 5A shows the result of RT-PCR analysis of expression of GAPDH,IL-2, IFN-γ, IL-4, IL-5, IL-10, TGF-β and iNOS mRNA following contact ofCD4⁺,CD25⁺ T cells, CD4⁺,CD25⁻ T cells, and combined CD4⁺,CD25⁻ T cellsand CD4⁺,CD25⁻ T cells from DA rats, with DA or PVG antigen. In thisassay duplicate samples at maximum concentration of cDNA were examinedas there is limited mRNA extracted from these cultures. CD4⁺,CD25⁺ Tcells alone cultured with self (DA) stimulators, have minimal cytokineinduction. With PVG stimulators there is no IL-2 mRNA but some IFN-γ,IL-4, IL-10 and TGF-β expression. In contrast the CD4⁺,CD25⁻ T cellshave marked induction of all cytokine mRNA tested when stimulated byPVG, and this profile is similar to that observed when stimulated withDA antigen. With self-stimulators there is no IL-5 mRNA induction,however. It should be noted that the CD4⁺,CD25⁻ T cells have a markedproliferative response to self, known as the autologous MLC, and thusmarked induction of cytokine mRNA in the CD4⁺,CD25⁻ T cells respondingto self is consistent with a marked autologous proliferative response.

In the mixing experiment of CD4⁺,CD25⁻ T cells and naive CD4⁺,CD25⁻ Tcells at a 1:1 ratio there is total suppression of proliferation asdescribed before. In these cultures there is marked reduction inexpression of IL-2, IL-4 and IL-5 mRNA in the allogeneic responsecompared to that with the CD4⁺,CD25⁻ T cells. There was a smallreduction in IFN-γ, IL-10, TGF-β mRNA and no effect on iNOS mRNA levelscompared to those observed with CD4⁺,CD25⁻ T cells alone against PVG.CTL are assays with cDNA from ConA stimulated T cells.

These results show that the suppression of MLC by the CD4⁺,CD25⁺ T cellsis associated with IFN-1 and iNOS induction, suggesting NO productionmay mediate this effect of suppression. This possibility was examined byadding an iNOS inhibitor L-NIL to MLC.

FIG. 5B

FIG. 5B is graphs showing the effect of blocking IL-5, TGF-β, IL-10 andiNOS on the inhibitory effects of CD4⁺,CD25⁺ T cells on CD4⁺,CD25⁻ Tcells proliferation in MLC.

In this experiment it was examined if iNOS or monoclonal antibodies toIL-5, TGF-β and IL-10 could block the suppression of CD4⁺,CD25⁺ T cellson CD4⁺,CD25⁻ T cells proliferation when they are cultured in a ratio of1:1. As can be seen in the bottom panel, the controls (CTN) are totallysuppressed and enhanced proliferation occurs to fully allogeneic donorwhen iNOS, anti-IL-5, anti-TGF-β and anti-IL-10 monoclonal antibody isadded but not with the control anti-Mog Ig2a monoclonal antibody. Nonerestored proliferation to that of CD4⁺,CD25⁻ T cells alone (see topright panel) and the effect was not non-specific as neither iNOS nor theantibodies had an effect on the proliferation of CD4⁺,CD25⁻ T cellsalone or on CD4⁺,CD25⁺ T cells alone, except for anti-TGF-β which didenhance CD4⁺,CD25⁺ T cells proliferation. These results suggest thatIL-5, IL-10 and TGF-β have a functional effect in the suppressiveeffects of CD4⁺,CD25⁺ T cells on CD4⁺,CD25⁻ T cells in MLC. Thissuggests that the non-specific suppression of CD4⁺,CD25⁺ T cells mayalso be in part mediated through the effects of IL-5 and IFN-γ.

Conclusions

The suppressive effect of naïve CD4⁺,CD25⁺ T cells on naïve CD4⁺, CD25⁻Tcells in MLC is associated with:

-   -   a) Naive CD4⁺,CD25⁺ T cells when admixed 1:1 with naïve        CD4⁺,CD25⁻ T cells totally suppress the MLC proliferation.    -   b) A marked reduction in induction of IL-2, IL-4, IL-5 mRNA        expression and a smaller reduction in induction of IFN-γ and        TGF-β mRNA expression, but no reduction in iNOS induction when        compared with the induction in CD4⁺,CD25⁻ T cells alone in MLC.    -   c) Blocking with anti-IL-5 or anti-TGF-β or anti-IL-10        monoclonal antibodies partially blocks suppression suggesting        IL-5, IL-10 and TGF-β have an effect in mediating the        suppression by naïve CD4⁺,CD25⁺ T cells on CD4⁺,CD25⁻ T cells        proliferation in MLC.

Example 6

The effect of IL-4 or IL-5 on survival in culture of CD4⁺ T cells fromDA rats with tolerance to PVG allografts was examined. The survival oftolerance mediating cells was assayed by their ability to adoptivelytransfer specific tolerance to PVG cardiac allografts and to retain thecapacity to effect rejection of third party Lewis allografts.

These studies used CD4 T cells from spleen and lymph nodes of DA ratstolerant to a PVG cardiac allograft that has survived over 75 days.Graft acceptance and tolerance developed after a short course ofimmunosuppression at the time of transplantation, either 10 days ofcyclosporine treatment, or anti-CD4 or anti-CD3 monoclonal antibodytherapy, as described (Transplantation, 1997, 64, 1559-1567 and 1993,55; 459-468). The ability of these cells to transfer tolerance wasexamined in the irradiated adoptive host that is described above inFIGS. 3A and 3B.

In early studies we had demonstrated that fresh tolerant cells transfertolerance to specific donor (PVG) but reject third party grafts.Admixing tolerant cells with naïve CD4⁺ T cells in a ratio of 4:1, ie 20million tolerant CD4⁺ T cells with 5 million naïve CD4⁺ T cells is usedto show the tolerant cells can suppress the naïve cells from effectingrejection. Previous studies and controls for these studies showed thatthe culture of tolerant CD4⁺ T cells with specific donor stimulators ina mixed lymphocyte culture for 3 days, resulted in their loss ofcapacity to transfer tolerance. Culture of naïve cells under similarconditions resulted in cells that effected rejection.

When tolerant CD4⁺ T cells were cultured with specific donor stimulatorsand with a cytokine rich media of ConA supernatant, then they retainedtheir capacity to transfer tolerance to specific donor but retained thecapacity to effect third party graft rejection as described(Transplantation 1993, 55, 374-379). This baseline data was reproducedas controls to the experiments and was as per the previous describedresults.

FIG. 6A

FIG. 6A shows the proliferation of CD4⁺ T cells from DA rats tolerant toa PVG heart allograft after being cultured with either IL-4 or IL-5alone in MLC with PVG antigen and no ConA supernatant. This experimentdemonstrated that IL-5 alone sustained the tolerance transferringspecific suppressor CDe T cells. The same cells retained the capacity toeffect third party graft rejection, demonstrating that the IL-5 alonedid not induce tolerance mediating cells. Neither IL-2 (Transplantation1993, 55, 374-379) nor IL-4 (see data above) alone maintain thesesuppressor CD4⁺ T cells in culture with specific antigen, while a Con Asupernatant with all these cytokines maintains the specific suppressorfunction of CD4⁺ T cells.

Taken together these experiments with culture of CD4⁺ T cells from ratswith allograft tolerance demonstrated that IL-5 alone can substitute forthe ConA supernatant. Either IL-2 or IL-4 alone is insufficient tomaintain the tolerance maintaining cells.

FIG. 6B

FIG. 6B illustrates the results of examination of the effect of culturein MLC with IL-4 and specific donor antigen on the capacity of tolerantCD4⁺,CD25⁺ T cells and CD4⁺,CD25⁻ T cells to proliferate in vitro whenexposed to different stimulator cells.

CD4⁺,CD25⁺ T cells from DA rats tolerant to a PVG cardiac allograft werecultured against PVG (specific allogeneic) stimulator cells in a primaryMLC with or without IL-4 for 3 days. These cells were subsequentlywashed, rested for 24 hours and then cultured in a secondary MLC aloneor against DA (syngeneic), PVG (specific allogeneic) or Lewis (3rd partyallogeneic) stimulator cells. Proliferation was assessed at day 2-3 ofsecondary culture.

The tolerant CD4⁺,CD25⁺ T cells have significantly reduced proliferationagainst specific donor PVG whether or not IL-4 is added to the primaryMLC. The CD4⁺,CD25⁻ T cells had a similar response to donor and thirdparty stimulators. These data are consistent with the findings in theadoptive transfer assay where IL-4 did not maintain the suppressoreffect of unfractionated CD4⁺ T cells, suggesting the specificallytolerant CD4⁺,CD25⁺ T cells die in culture if IL-4 is the only cytokineavailable. Also that the CD4⁺,CD25⁻ T cells cultured with IL-4 surviveand maintain alloreactivity to specific donor and third party. Theseresults are consistent with the adoptive transfer studies described inFIG. 6A where tolerant cells cultured with IL-4 do not maintain theirability to transfer specific tolerance. This study also demonstratesthat the culture of specific CD4⁺,CD25⁻ T cells from tolerant animalscannot be maintained by IL-4 and IL-4 does not reinduce tolerance. Thatis activated CD4⁺,CD25⁺ T cells from tolerant animals are not maintainedor expanded by the IL-4 and alloantigen.

Conclusion

The Th2 cytokine IL-5 but not IL-4, promotes the survival of specificCD4⁺,CD25⁺ T cells tolerance mediating suppressor cells in culture andallows them to retain the ability to transfer tolerance.

-   -   a) Survival of the alloantigen specific tolerance mediating        cells in CD4⁺ T cells in MLC can be supported by IL-5 or Con A        sup. IL-4 supplement alone does not promote survival of the        tolerance mediating CD4⁺ T cells.    -   b) The failure of tolerant CD4⁺,CD25⁺ T cells to proliferate to        specific donor in MLC is not restored by pre-culture with        specific donor alloantigen and IL-4.

Example 7

This study examined the effect of various cytokines on proliferation ofactivated CD4⁺,CD25⁺ T cells in MLC.

FIG. 7

FIG. 7 examines the effect of individual cytokines on proliferation inMLC of unfractionated lymphocytes from DA rats with tolerance to PVGheart allografts.

In this experiment the effects of various cytokines on proliferation ofunfractionated lymphocytes from DA rats with tolerance to a PVGallograft were examined MLC. The proposal was that key cytokines wouldpromote survival of the activated CD4⁺,CD25⁺ T cells that maintaintolerance and these would inhibit the proliferative response ofunfractionated cells in MLC.

This figure shows IL-2 and IL-4 markedly enhance proliferation to selfDA and to specific-donor PVG. Both IL-5 and IFN-γ inhibit responses toPVG but not self. Other cytokines had a similar effect, including IL-10,IL-12p40 homodimer, and IL-12p70.

This data suggests the test for the tolerant state may be detected byadding IL-5, IL-12 or IFN-γ. These cytokines may inhibit proliferationof unfractionated tolerant lymphocytes by promoting the survival andfunction of the donor antigen specific activated CD4⁺,CD25⁺ T cells sothey suppress the proliferative response in MLC. This effect may bedetected by decreased proliferation or by other means that detect activefunction of the activated CD4⁺,CD25⁺ T cells.

Conclusion

Addition of cytokines that promote the survival of activated CD4⁺,CD25⁴T cells from tolerant animals reduces the proliferation ofunfractionated peripheral lymphocytes to specific donor antigen in MLC,in particular IL-5, IFN-γ and IL-12.

Example 8

This study examined the ability to treat EAN in rats by administeringactivated CD4⁺,CD25⁴′ T cells.

Methods Methods;

EAN was induced in 10-15 week old female Lewis rats by immunization withbovine peripheral nerve myelin in Freund's complete adjuvant, asdescribed (J. Neurol. Sci. 1994, 123: 162-172). The animals weremonitored for disease activity daily by weighing and clinicalobservation and scoring of paralysis using a semi-quantitative score.The score used was; 5+ death or total paralysis requiring euthanasia, 4+paralysis of all limbs, 3+ Total hind limb paralysis, and weak forearms,2+ weak hind limbs, 1+ weak tails, 0 normal.

Results FIG. 8

In FIGS. 8A and 8B, the effect of adoptive transfer of activatedCD4⁺,CD25⁺ T cells or CD4⁺,CD25⁻ T cells on the clinical course ofExperimental Allergic Neuritis (EAN) in Lewis rats was examined.

As proof of concept, the effect of CD4⁺,CD25⁺ T cells from Lewis ratsthat had recovered from EAN on the development of EAN in naïve rats wasexamined by adoptive transfer of these cells to naïve Lewis rats at thetime of their immunization with PNM in Freund's complete adjuvant. Theeffect of these tolerant CD4⁺,CD25⁺ T cells was compared to the effectof CD4⁺,CD25⁺ T cells from these tolerant animals.

In this model rats that recover, appear normal and are resistant tore-induction of EAN by re-immunization with immunogenic antigen. That isthey are considered tolerant after their original disease, thus ratsthat had just recovered from EAN were considered a good source ofactivated tolerant CD4⁺,CD25⁺ T cells. Lewis rats that had recoveredfrom EAN at 30 days post immunization were thus used to prepareCD4⁺,CD25⁻ T cells and CD25⁺,CD4⁺ T cells. All groups of Lewis rats(n=5-12) were immunized with peripheral nerve myelin (PNM) and Freund'sadjuvant. One group was given 5 million CD25⁺,CD4⁺ T cells ivi andanother group was given 5 million CD4⁺,CD25⁻ T cells ivi. The controlgroup was not given any cells at the time of immunization. Those givenactivated CD4⁺,CD25⁺ T cells from tolerant rats had a much milderclinical course with a maximum disease score just over one, compared tocontrols whose diseases peaked at 2.5+ around 15-16 days postimmunization. Rats given CD4⁺,CD25⁻ T cells from tolerant animalsdeveloped more severe disease peaking at 3+ and with an earlier onsetand slower recovery.

In another experiment naïve CD4⁺,CD25⁺ T cells were given on day 0 ofimmunization. These had no effect on the clinical course of EANconfirming this was an effect of activated CD4⁺,CD25⁺ T cells from atolerant animal.

Weight loss was much less in those given activated CD4⁺,CD25⁺ T cellsfrom a tolerant animal and slightly greater in those given CD4⁺,CD25⁻ Tcells from a tolerant animal than controls given no cells. (FIG. 8B)

As adult rats have 500-1000 million peripheral lymphocytes, we suggestthat 5 million CD4⁺,CD25⁺ T cells would not have had a significantimpact of the CD25⁺,CD4⁺ T cell: CD4⁺,CD25⁻ T cell ratio in these naïvehosts. This is further supported by the finding that giving 5 millionnaïve CD4⁺,CD25⁺ T cells had no effect on the course of EAN. Thus theeffect was most likely due specifically to the specifically activatedCD4⁺,CD25⁺ T suppressor cell effect, not the well described non-specificeffect of naïve CD4⁺,CD25⁺ T cells.

This suggests a finite number of activated CD4⁺,CD25⁺ T cells maysignificantly alter the disease course and re-establish tolerance,during an acute disease process.

Conclusions

Specific activated CD4⁺,CD25⁺ T cells from animals that have recoveredfrom acute autoimmune diseases can ameliorate the severity of autoimmunedisease.

We propose that autologous CD4⁺,CD25⁺ T cells may be obtained fromdiseased animals in remission and further activated to transfer back tomaintain or re-establish tolerance.

Example 9

Cell subsets and cultures were as per the mixed lymphocyte cultures.Antigen presenting cells with PNM were prepared by pre-culturing for 1hours at 37 degrees with PNM and the autologous stimulator cellpreparation. These cells were then washed. Culture conditions andmeasurement of proliferation was as described for MLC.

Results

These studies demonstrate distinct patterns of response of CD4⁺,CD25⁺ Tcells to an autoantigen before and during exposure to the autoantigen,which in this case was PNM in the EAN model.

First the response of naïve T cells, CD4⁺,CD25⁺ T cells and CD4⁺,CD25⁻ Tcells, showed that the CD4⁺,CD25⁻ T cells had a much greater responsethan the CD4⁺ T cells, and that the response of CD4⁺,CD25⁻ T cells couldbe inhibited by naïve CD4⁺,CD25⁺ T cells. Naïve CD4⁺,CD25⁺ T cells alonehad little or no response to PNM. This was similar to the ability ofnaïve CD4⁺,CD25⁺ T cells to inhibit naïve CD4⁺,CD25⁻ T cells in MLC.(FIG. 9A)

Second in animals that are just recovering there is an active suppressorphenotype, where the CD4⁺,CD25⁺ T cells do not greatly respond tospecific antigen and cannot fully inhibit the response of CD4⁺,CD25⁻ Tcells to the specific antigen. We would predict that these cells mayrespond to IL-5, IFN-γ and possibly IL-12. (FIG. 9B)

FIG. 9A.

Referring to FIG. 9A, proliferation of lymphocytes from naïve Lewis ratswhen stimulated in culture with self-antigen presenting cells with andwithout PNM was examined. Proliferation at day 4 vs day 5.

The proliferation of naïve Cpe,CD25⁺ T cells and CD4⁺,CD25⁻ T cells tothat of naïve unfractionated CDT″ T cells was examined. This response issimilar to that observed with naïve cells response to alloantigens inMLC. At day 4 the response of unfractionated CD4⁺ T cells was similar toself and self plus PNM. That of CD4⁺,CD25⁻ T cells was greater thanunfractionated CD4⁺ T cells, consistent with removal of the non-specificsuppression by naïve CD4⁺,CD25⁺ T cells. Again there was no differencein the response to self alone and self plus PNM. CD4⁺,CD25⁺ T cells hadminimal proliferation alone, and when admixed with CD4⁺,CD25⁻ T cells ina normal ratio, suppressed the response to that of unfractionated CD4⁺ Tcells. With a ratio of 1:1 naïve CD4⁺,CD25⁺ T cells: naïve CD4⁺,CD25⁻ Tcells there was near total suppression.

The results on day 5 had an essentially similar pattern but there wasgreater proliferation than on day 4. Again admixing the two populationsat 1:1 ratio markedly suppressed the response to both self alone andself with PNM.

FIG. 9B

Referring to FIG. 9B, in this study T cell subsets were prepared fromLewis rats that had recovered from EAN, 30 days after immunization.Again culture stimulated with self antigen presenting cells alone orprimed with PNM were set up and proliferation assayed at days 4 and 5.The response of unfractionated CD4⁺ T cells and CD4⁺,CD25⁻ T cells wasgreater to PNM than to self-antigen presenting cells, consistent with aspecific sensitisation. The CD4⁺,CD25⁺ T cells alone had a non-specificproliferation above that normally seen with naïve CD4⁺,CD25⁺ T cells.This may be due to polyclonal activation of the CD4⁺,CD25⁺ T cells.

The major difference was that the suppression with CD25⁺,CD4⁺ T cells at1:1 against PNM was incomplete, whilst that to self was more complete.Our interpretation of the results of this assay is that there are tworesponses, that to self-antigens and that to PNM. The data is consistentwith partial suppression of the anti-self response but failure tosuppress that anti-PNM response. These data are similar to that observedin transplant tolerance in that the activated CD4⁺,CD25⁺ T cells whichhave alloantigen specificity do not suppress the response to thespecific antigen, but retain their capacity to non-specifically inhibita third party response.

Conclusions

That CD4⁺,CD25⁺ T cells from naïve animals behave in autoimmuneresponses like they behave with alloimmune responses.

-   -   a) That in naïve animals there is no specific response to        autoantigen, in that they are the same as that to self        stimulators.    -   b) That in naïve animals removal of CD4⁺,CD25⁺ T cells enhances        the response to self stimulators. That is naïve CD4⁺,CD25⁻ T        cells proliferation is greater than that of unfractionated CD4⁺        T cells.    -   c) Further naïve CD4⁺,CD25⁻ T cells proliferation can be        suppressed by admixing with CD4⁺,CD25⁺ T cells, especially at a        1:1 ratio.    -   d) That in animals recovering from acute episode of        autoimmunity, there is an enhanced response to specific antigen        compared to self antigens, for CD4⁺ T cells and CD4⁺,CD25⁻ T        cells, but no specific response by CD4⁺,CD25⁺ T cells to the        autoantigen. These CD4⁺, CD25⁺ T cells do not fully suppress the        CD4⁺,CD25⁻ T cells proliferation, and are more effective against        the self response than against the specific alloantigen. That is        the activated specific CD4⁺,CD25⁺ T cells do not suppress in        vitro.

Example 10

This example illustrates the ability of CD4⁺,CD25⁺ T cells cultured invitro in the presence of donor antigen and IL-2 or IL-4 to suppressdonor heart graft rejection.

Ts1 cells (activated CD4⁺,CD25⁺ T cells produced by alloantigenstimulation in the presence of IL-2, expressing IFNGR and IL-12Rβ2) andTs2 cells (activated CD4⁺,CD25⁺ T cells produced by alloantigenstimulation in the presence of IL-4, expressing IL-5Rα and IFN-γ) wereproduced by 3 day culture of naïve CD4⁺,CD25⁺ T cells from DA rats inthe presence of antigen presenting cells from PVG rats and >100 units/mlof IL-2 or IL-4 respectively. These cells were then tested for theircapacity to prevent PVG or third party cardiac allograft rejection inirradiated DA rats restored with 5×10⁶ naïve CD4⁺ T cells. The resultsof the experiment are shown in Table 2.

As can be seen from Table 2, naïve CD4⁺,CD25⁺ T cells in a ratio of 1:10with naïve CD4⁺ T cells did not suppress rejection of hearts from PVGdonor strains. Similarly, CD4⁺,CD25⁺ T cells contacted with PVG antigenin the presence of IL-2 or IL-4 and mixed in a ratio of 1:10 with CD4⁺ Tcells did not suppress rejection of hearts from Lewis donor strains. Incontrast, CD4⁺,CD25⁺ T cells contacted with PVG antigen and incubated inthe presence of IL-2 or IL-4 and mixed in a ratio of 1:10 with naïveCD4⁺ T cells did suppress rejection of hearts from PVG donor strains.Thus, both Ts1 and Ts2 cells at a ratio of 1:10 (TS1 or TS2 T cells toCD4⁺ T cells) prevented rejection of PVG but not Lewis allografts,demonstrating specificity of induction of suppression and enhancedsuppression as naïve CD4⁺CD25⁺ T cells at ratio of 1:10 did notsuppress. Naïve CD4⁺CD25⁺ T cells at a ratio of 1:1 suppressed both PVGand Lewis rejection. Thus a short culture with specific alloantigen andeither IL-2 or IL-4 selected for and expanded specific suppressorCD4⁺CD25⁺ T cells leading to a 10-fold increase in suppressor capacity.

TABLE 2 Adoptive transfer* animals restored with Animals with severerejection^(†) CD4⁺, CD25⁺ cells Naïve CD4⁺ Heart donor Number/ Days posttransplant^(§) Significance No. Culture with cells rat strain TotalMedian Day (number of animals) (p)^(‡) — — 5 × 10⁶ PVG 12/12 12 10(3),12(4), 13, 14(2), 18, 20 — — — 5 × 10⁶ Lewis  3/3 10 9, 10(2) —   5 ×10⁶ — 5 × 10⁶ PVG  3/9 >100 16(3), >100(6) p = 0.0006 0.5 × 10⁶ — 5 ×10⁶ PVG  8/9 14 8, 13(3), 14(4), >100 NSD 0.5 × 10⁶ IL-2 and PVG cells 5× 10⁶ PVG  1/6 >100 12, >100(5) p = 0.0066 0.5 × 10⁶ IL-2 and PVG cells5 × 10⁶ Lewis  5/5 11 9, 11(4) NSD 0.5 × 10⁶ IL-4 and PVG cells 5 × 10⁶PVG  2/6 >50 10, 14, >50 (4) p = 0.0351 0.5 × 10⁶ IL-4 and PVG cells 5 ×10⁶ Lewis  4/6 19(13-24) 8, 9, 13, 24, >50 (2) NSD *In the adoptivetransfer assay, the recipient and donor are irradiated and a heart graftperformed one day later. The irradiated recipients do not reject theirgraft but rejection can be restored with naïve CD4⁺ T cells. ^(†)Severerejection refers to rejection associated with major swelling, loss ofcontraction and slowing of beat, equivalent to major graft dysfunction.Clinically this severity of rejection would be incompatible with life.^(§)Number of days post-transplantation at which severe rejectionoccurred for those animals which underwent rejection. Animals that didnot have a severe rejection episode had excellent heart graft functionfor >50-100 days. ^(‡)Compared to animals reconstituted with 5 × 10⁶naïve CD4⁺ T cells of the same heart donor strain. NSD; notsignificantly different

Example 11

These experiments examined whether CD4⁺,CD25⁺ T cells from DA ratsactivated to antigen from PVG rats and cultured in the presence of PVGcells and IFN-γ were capable of suppressing rejection of a PVG cardiacgraft in DA rats.

CD4⁺ T cells from DA rats tolerant to a cardiac allograft from PVG ratswere cultured in mixed lymphocyte culture with PVG alloantigen (asdescribed in Example 1) and >100 units/ml of IFN-γ. After three days theT cells were adoptively transferred to irradiated DA rats grafted witheither specific donor PVG cardiac allografts (grafts from PVG rats), orthird party Lewis allografts (grafts from Lewis rats). Each irradiatedrat was then restored with 5×10⁶ naïve CD4⁺ T cells. The survival ofgrafts was then monitored over a 50 day period. The results of theexperiment are shown in FIG. 10. The upper graph of FIG. 10 shows thesurvival when T cells were adoptively transferred to irradiated DA ratsgrafted with PVG cardiac allografts. The lower graph of FIG. 10 showsthe survival when T cells were adoptively transferred to irradiated DArats grafted with third party Lewis cardiac allografts.

As can be seen from FIG. 10, the only grafts which demonstratedsignificant survival over the 50 day period were grafts in rats whichreceived no cells (see FIG. 10 upper and lower graphs, solid line), orPVG grafts (but not Lewis grafts) in rats which received CD4⁺ T cellscultured in the presence of PVG antigen and IFN-γ (see FIG. 10, uppergraph, dotted line closed circles). The CD4⁺ T cells cultured with IFN-γwhen admixed with 5×10⁶ naïve CD4⁺ T cells suppressed allograftrejection, indicating that they retained suppressor capacity.

These results indicate that the cultured CD4+ T cells continued tosuppress PVG allograft rejection but did not suppress Lewis graftrejection. Naïve cells alone effected rejection, whereas irradiationdelayed rejection.

Thus, these results demonstrate that IFN-γ can preserve survival ofactivated CD4⁺,CD25⁺ T cells in a CD4⁺ T cell population from tolerantanimals.

Example 12

FIG. 11 illustrates an experiment that was conducted to compare theproliferation of unfractionated CD4⁺ T cells, a CD4⁺CD25⁻ T cell subsetand a CD4⁺CD25⁺ T cell subset from naïve DA rats after culturing the Tcells for 4 days in the presence of autoantigen (DA antigen) oralloantigen (PVG antigen) in media supplemented with IL-2 or IL-12p70 orboth IL-2 and IL-12p70. The effect of the antigen and cytokinecombination on cell proliferation is shown in FIG. 11. Proliferation ofunfractionated CD4⁺ T cells is shown in the top row of graphs,proliferation of the CD4⁺CD25⁻ T cell subset is shown in the middle rowof graphs, and proliferation of the CD4⁺CD25⁺ T cell subset is shown inthe lower row of graphs. The left hand column of graphs showproliferation in response to autoantigen, and the right hand column ofgraphs show proliferation in response to alloantigen (PVG).

As can be seen from FIG. 11, proliferation was not enhanced by IL-12p70alone when compared to control nil cytokines. IL-2 induced markedproliferation of all subsets, and addition of IL-12p70 enhanced thisproliferation in the CD4⁺ and the CD4⁺CD25⁺ populations but not in theCD4⁺CD25⁻ T cells.

These results indicate that IL-12p70 enhances proliferation ofCD4+,CD25+ T cells that have been activated with antigen and IL-2 (Ts1cells).

In parallel experiments, culturing of cells with IL-4 and IL-12p70 didnot enhance proliferation above that with IL-4 clone.

Example 13

This experiment examined the effect of IL-2 and IL-12p70 on growth ofTs1 cells (CD4⁺,CD25⁺ T cells activated by culturing in the presence ofantigen and IL-2).

Ts1 cells were prepared by culturing naïve CD4⁺CD25⁺ T cells in thepresence of IL-2 and alloantigen (PVG antigen) for 3 days. Cells werethen washed and place in fresh media with alloantigen and either nosupplement, CHO-K supernatant, IL-2, IL-12p70 or IL-12p40>100 units/ml.Cell proliferation was then measured as described above. The results ofcell proliferation are shown in FIG. 12.

As can be seen from FIG. 12, cultures with control CHO-K supernatant orIL-12p40 exhibited proliferation similar to those with no supplement.Culturing in the presence of IL-12p70 induced significant extraproliferation compared to controls, as did culturing in the presence ofIL-2. These experiments indicate that Ts1 cells are responsive toIL-12p70.

Example 14

Proliferation of unfractionated CD4⁺ T cells, a CD4⁺CD25⁺ T cell subsetand a CD4⁺CD25⁺ T cell subset from naïve DA rats was compared afterculture for 4 days against auto-antigen (DA antigen) or alloantigen (PVGantigen) when media was supplemented with no cytokine, IL-4 or IL-12p70or both IL-12 and IL-12p70. Proliferation was not enhanced by IL-12p70alone when compared to control nil cytokines. IL-4 induced markedproliferation of all subsets, and addition of IL-12p70 did not enhanceproliferation of any of the subpopulations. This showed IL-12p70 did notenhance proliferation of Ts2 cells.

Example 15

RT-PCR was used to analyse the expression of IL-2 and IL-12β2 receptormRNA in CD4⁺,CD25⁺ T cells from naïve DA rats following culturing withalloantigen (cells from PVG rats) or autoantigen (cells from DA rats)and IL-2 or IL-4>100 units/ml. The results of the RT-PCR analysis areshown in FIG. 13.

FIG. 13 illustrates RT-PCR of GAPDH (control), IL-2 or IL-12132 receptormRNA following culture will alloantigen (PVG) or autoantigen (DA) for 4days with no cytokine (upper panels); IL-2, (middle panel); or IL-4(lower panel). As can be seen from FIG. 13, strong bands were observedfor IL-142 receptor when cells were cultured with IL-2 and alloantigen.A feint band was observed for IL-12β2 receptor following alloantigenstimulation alone. This result suggests preferential up regulation ofthe IL-12 receptor on CD4⁺CD25⁺ T cells activated by contacting withalloantigen in the presence of IL-2.

Example 16

EAN was induced in 10-15 week old female Lewis rats by immunization withbovine peripheral nerve myelin (PNM) in Freund's complete adjuvant, asdescribed in J. Neurol. Sci. 1994, 123: 162-172. The animals weredivided into three groups: (a) those immunised with PNM and Freund'sadjuvant only (control); (b) those immunised with PNM and Freund'sadjuvant and administered CHO cell supernatant (control); and (c) thoseimmunised with PNM and Freund's adjuvant and administered IL-5 (5000units/day daily intraperitoneal injection from the day of onset ofclinical for 10 days).

All groups of Lewis rats (n=5-12) were immunised with peripheral nervemyelin (PNM) and Freund's adjuvant as described above.

The animals were monitored for disease activity daily by weighing andclinical observation and scoring of paralysis using a semi-quantitativescore. The score used was; 4+ paralysis of all limbs, 3+ Total hind limbparalysis, and weak forearms, 2+ weak hind limbs, 1+ weak tails, 0normal.

Referring to FIG. 14A, the effect of administration of IL-5 on theclinical course of Experimental Allergic Neuritis (EAN) in Lewis ratswas examined.

As can be seen from FIG. 14A, those rats administered IL-5 had a milderclinical course with a maximum disease score just over one, compared tocontrols whose diseases peaked at 2.5+ around 15-16 days postimmunization.

FIG. 14B illustrates weight loss over the course of the disease. Weightloss was less in those rats administered IL-5 when compared to theuntreated control of following treatment with CHO cell supernatant.

The percent demyelination was also investigated in rats immunised withPNM with or without IL-5. The effect of treatment with IL-5 ondemyelination at day 14 and 21 is shown in FIG. 15. As can be seen fromFIG. 15, treatment with IL-5 may reduce the demyelination normallyobserved in the EAN model.

The above data suggests that administration of IL-5 may be effective inreducing the severity of EAN by inducing tolerance to PNM.

Example 17

To determine the expression of cytokines in animals immunised with PNMand treated with IL-5 as described in Example 16, real time PCR analysisof mRNA from draining lymph nodes (LN) and cauda equina (CE) wasperformed at day 14 and 21 post immunisation with PNM. The results ofthe real-time PCR is shown in FIG. 16. Open bars represent mRNA levelsof control rats that were not treated with IL-5, while closed barsrepresent mRNA levels of rats treated with IL-5. Cytokines that weremeasured are as indicated at the top of the diagram.

As can be seen from FIG. 16, in the lymph node draining the site ofimmunization, there was a marked increase in IFN-γ expression but notIL-2 in IL-5 treated rats (closed bars) at day 21 compared to controls(open bars), whereas IL-10 was reduced at day 14. In the cauda equina,there was marked reduction in IL-2 and IFN-γ, indicating reduced Th1cell infiltration. TCR-α was less in both the lymph nodes and caudaequina at day 21 but not day 14. TCR-α, IL-10 and TNF-α results areexpressed as copy number/100000 GAPDH copies. IL-2 and IFN-γ results areexpressed as copy number/100 TCR-α copies. Data was combined ofduplicate assays from 5 samples, expressed as mean±SD. Statisticalsignificant differences *p<0.05, **p<0.005.

Example 18

Expression of IL-5, IL-5 receptor, IL-4 and IL-13 in draining lymphnodes and cauda equine in rats that had been immunised with PNM and wereeither untreated (CTL) or treated with IL-5 as described in Example 15were examined using semi-quantitative RT-PCR. The results ofsemi-quantitative RT-PCR are shown in FIG. 17.

Referring to FIG. 17, semi-quantitative RT-PCR of cytokine mRNA showedIL-5 treated animals have increased IL-5 and IL-5Ra in the lymph nodedraining the site of immunization. There was similar expression of IL-4but reduced IL-13 in IL-5 treated compared to control (CTL). In thecauda equina, IL-5 and IL-5Ra was still detected, even though TCR-αcopies were reduced (refer to FIG. 3). Each box shown serial cDNAdilutions with neat on right and 1:10 dilution on the left. ControlGAPDH expression was similar in all samples. Results from 5 samples pergroup with one representative sample shown.

Example 19

The effect of naïve and activated CD4⁺CD25⁺ T cells on proliferation ofnaïve CD4⁺CD25⁻ T cells in MLC was tested in a limiting dilution assaywith a 1:2 serial dilutions of CD4⁺,CD25⁺ T cells were mixed with aconstant number of naïve (10⁵) CD4⁺,CD25⁻ T cells. The results of thelimiting dilution study are shown in FIG. 18.

FIG. 18(A) shows the ability of fresh naïve CD4⁺CD25⁺ T cells topartially suppress responses to PVG antigen and Lewis antigen, at aratio of 1:1. The ability of the naïve T cells to suppress response toPVG antigen is significant diminished at a ratio of 1:8 (naïveCD4⁺,CD25⁺CD4⁺,CD25⁻ T cells).

FIG. 18(B) shows Ts 1 cells (CD4⁺,CD25⁺ T cells activated to antigen (inthis case PVG antigen in the presence of IL-2) selectively fullysuppress responses to PVG antigen to a ratio of 1:16 but only suppressto Lewis antigen (third party) at a ratio of 1:4 then lose suppression.

Referring to FIG. 19A, FIG. 19A shows that fresh naïve CD4⁺CD25⁺ T cellspartially suppress responses to PVG antigen and Lewis antigen, at aratio of 1:1 but loose significant inhibition at a ratio of 1:8. FIG.19(B) shows that Ts2 cells (CD4⁺,CD25⁺ T cells activated to (in thiscase PVG antigen) in the presence of IL-4) partially suppress bothresponses to PVG and Lewis antigen and do so in ratios similar to thatobserved with fresh naïve cells. In other studies we have shown the MLCmainly induces Th1 responses with induction of IL-2 and IFN-γ. The onlyTh2 cytokine induced is IL-4 with no IL-5 or IL-10 unless cells areexposed to extra IL-4, when IL-2 and IFN-γ induction is significantlysuppressed an IL-4 and IL-5 expression enhanced.

Example 20

This experiment was conducted to determine the affect of incubatingCD4⁺,CD25⁺ T cells in the presence of IL-23 and IL-13 followingactivation of naïve CD4⁺,CD25⁺ T cells in the presence of IL-2 or IL-4.

Naïve CD4⁺,CD25⁺ T cells from naïve DA rats were incubated withstimulator cells from PVG rats in the presence of either IL-2 (group A)or IL-4 (group B) for 3 to 4 days as described above. Following 3 to 4days, the cells were washed and the culture medium replaced with culturemedium containing either 100 units/ml IL-12p70 (positive control),IL-23, IFN-γ, IL-10 or IL-12p70 and IFN-γ for group A, and 100 units/mlIL-13 or IL-13 and IL-5 for group B. Negative control was CHO cellsupernatant.

CD4⁺,CD25⁺ T cells incubated in the presence of IL-12p70, IL-23 andIL-13 alone exhibited further proliferation. CD4⁺,CD25⁺ T cellsincubated in the presence of IL-13 and IL-5, or IL-12p70 and IFN-γ alsoshowed enhanced proliferation.

The following examples are prophetic examples intended to illustratevarious embodiments of various aspects of the invention.

Prophetic Example 1

This example illustrates the use of CD4⁺,CD25⁺ T cells to determinewhether a subject is tolerant to a specific antigen.

CD4+,CD25+ T cells may be isolated from lymphocyte cultures obtainedfrom patients in which tolerance to a specific antigen is to be tested.The CD4⁺,CD25⁺ T cells may be isolated using the methods describedabove.

In one form, aliquots of approximately 10⁵ CD4⁺,CD25⁺ T cells from asubject are contacted with donor antigen, self antigen or a third partyantigen in culture medium that is cytokine free or does not containIL-2, IL-4, IL-5, IL-12 or IFN-γ for at least 3 days, typically 5 days.Cell proliferation after 3 days is measured as described above. FIGS.20(A) and (B) illustrates the expected result from a subject that istolerant (A) or non-tolerant (B) to the donor antigen. As can be seen inFIG. 20, there will be little or no proliferation of CD4⁺,CD25⁺ T cellsfrom a subject that is tolerant to the donor antigen (A) in the absenceof IL-5, IL-12 or IFN-γ. In this regard, cell proliferation ofCD4⁺,CD25⁺ T cells in contact with donor antigen would be comparable toproliferation of CD4⁺,CD25⁺ T cells in contact with self antigen. Incontrast, CD4⁺,CD25⁺ T cells from a non-tolerant subject (B) willexhibit similar proliferation as that observed in response to contactwith third party antigen.

In another form, aliquots of approximately 10⁵ CD4⁺,CD25⁺ T cells from asubject are contacted with donor antigen, self antigen or a third partyantigen, in culture medium such as RPM1 with 10% FCS that issupplemented with 10 mg/ml (HOW MUCH?) IL-5, IFN-γ and/or IL-12p70 forat least 3 days. Cell proliferation after 3 days is measured asdescribed above. FIGS. 20C and D illustrate the expected result from asubject that is tolerant (C) or non-tolerant (D) to the donor antigen.As can be seen in FIG. 20, CD4⁺,CD25⁺ T cells from a subject that istolerant to the donor antigen would proliferate in the presence of IL-5,IL-12 or IFN-γ. In this regard, cell proliferation of CD4⁺,CD25⁺ T cellsin contact with donor antigen would be greater than proliferation ofCD4⁺,CD25⁺ T cells in contact with third party antigen. In contrast,CD4⁺,CD25⁺ T cells from non-tolerant subjects in contact with donorantigen would show similar proliferation as that observed for cells incontact with third party antigen.

Prophetic Example 2

This example illustrates the use of unfractionated CD4⁺ T cells todetermine whether a subject is tolerant to a specific antigen.

In one form, aliquots of approximately 10⁵ CD4⁺ T cells from a subjectare contacted with donor antigen, self antigen or third party antigen inculture medium (such as RPM1 supplemented with 10% FCS) that is cytokinefree or is at least not supplemented with IL-2, IL-4, IL-5, IFN-γ orIL-12p70 for at least 4 days. Cell proliferation after 4 days ismeasured as described above. FIGS. 21(A) and (B) illustrates theexpected result from a subject that is tolerant (A) or non-tolerant (B)to the donor antigen. Proliferation in response to donor antigen will besimilar to that of the response to the third party antigen due to deathof CD4⁺,CD25⁺ T cells in the absence of cytokine and therefore inabilityto suppress CD4⁺,CD25⁻ T cells, as well as reduced numbers of CD4⁺,CD25⁻T cells, as indicated in FIG. 1F.

Referring to FIG. 21(B), in a non-tolerant subject, the response todonor antigen will be the same as that to third party antigen as bothhave normal numbers of CD4⁺,CD25⁺ T cells, and suppression by naïveCD4⁺,CD25⁺ T cells.

In the presence of IL-5, IL-12, or IFN-γ, activated CD4⁺,CD25⁺ T cellsfrom tolerant subjects will survive and suppress the proliferation ofCD4⁺CD25⁻ T cells in response to donor antigen as illustrated in C.Thus, proliferation of CD4⁺ T cells from tolerant subjects in responseto donor antigen will be reduced and similar to the response to self andless than the response to third party antigen. CD4⁺ T cells fromnon-tolerant individuals (see FIG. 21D) will exhibit a similarproliferation to donor antigen as that in response to third partyantigen.

In the claims which follow and in the preceding description of theinvention, except where the context requires otherwise due to expresslanguage or necessary implication, the word “comprise” or variationssuch as “comprises” or “comprising” is used in an inclusive sense, i.e.to specify the presence of the stated features but not to preclude thepresence or addition of further features in various embodiments of theinvention.

1-65. (canceled)
 66. A method of increasing tolerance to a specificantigen in a subject in need thereof, comprising administering to thesubject an effective amount of CD4⁺,CD25⁺ T cells activated to thespecific antigen grown in vitro by culturing CD4⁺,CD25⁺ T cellsactivated to the specific antigen in the presence of at least onecytokine selected from the group consisting of IL-5, IL-12, IL-23 andIFN-γ or a biologically active fragment thereof, or a functionallyequivalent molecule thereof.
 67. The method of claim 66, wherein theCD4⁺,CD25⁺ T cells activated to the specific antigen are contacted withthe specific antigen in vitro prior to, or simultaneous with, culturingthe activated T cells in the presence of the at least one cytokine. 68.The method of claim 66, further comprising administering to the subjectone or more antibodies which specifically bind to CD4⁺,CD25⁻ T cells.69. The method of claim 66, wherein the CD4⁺,CD25⁺ T cell activated tothe specific antigen are activated to the specific antigen in vitro bycontacting naïve CD4⁺,CD25⁺ T cells with the specific antigen in vitro,and culturing the CD4⁺,CD25⁺ T cells in the presence of one or morecytokines capable of supporting activation of CD4⁺,CD25⁺ T cells. 70.The method of claim 69, wherein the one or more cytokines capable ofsupporting activation of CD4⁺,CD25⁺ T cells are selected from the groupconsisting of IL-2, a biologically active fragment thereof, or afunctionally equivalent molecule thereof, and IL-4, a biologicallyactive fragment thereof, or a functionally equivalent molecule thereof.71. The method of claim 66, wherein the CD4⁺,CD25⁺ T cells activated tothe specific antigen are activated to the specific antigen in vivo priorto culturing in vitro.
 72. The method of claim 66, wherein a mixture ofnaïve and activated CD4⁺,CD25⁺ T cells are grown by contacting naïve andactivated CD4⁺,CD25⁺ T cells with the specific antigen in vitro, andculturing the naïve and activated CD4⁺,CD25⁺ T cells in the presence ofIL-2, a biologically active fragment thereof, or a functionallyequivalent molecule thereof, and/or IL-4, a biologically active fragmentthereof, or a functionally equivalent molecule thereof, and at least onecytokine selected from the group consisting of IL-5, IL-12, IL-23 andIFN-γ, or a biologically active fragment thereof, or a functionallyequivalent molecule thereof.
 73. The method of claim 66, wherein the atleast one cytokine has been added exogenously to medium in which theCD4⁺,CD25⁺ T cells are cultured.
 74. The method of claim 66, furthercomprising administering to the subject an effective amount of at leastone cytokine selected from the group consisting of IL-5, IL-12, IL-23and IFN-γ or a biologically active fragment thereof, or a functionallyequivalent molecule thereof, simultaneously with, or subsequent to,administering the activated CD4⁺,CD25⁺ T cells.
 75. The method of claim66, wherein the specific antigen is autoantigen, an alloantigen, axenoantigen, a tumour antigen, an allergen, or an antigen from aninfectious agent.
 76. A method for treating or preventing in a subjectin need thereof a disease resulting from an immune response to aspecific antigen, the method comprising administering to the subject atherapeutically effective amount of CD4⁺,CD25⁺ T cells activated to thespecific antigen grown in vitro by culturing CD4⁺,CD25⁺ T cellsactivated to the specific antigen in vitro in the presence of at leastone cytokine selected from the group consisting of IL-5, IL-12, IL-23and IFN-γ, or a biologically active fragment thereof, or a functionallyequivalent molecule thereof.
 77. The method of claim 76, wherein theCD4⁺,CD25⁺ T cells activated to the specific antigen are contacted withthe specific antigen in vitro prior to, or simultaneous with, culturingthe activated CD4⁺,CD25⁺ T cells in the presence of the at least onecytokine.
 78. The method of claim 76, wherein the disease is: (a) adisease associated with an immune response to an autoantigen; (b) adisease that is the result of an immune response to a non-self antigenin contact with the subject; or (c) a disease that is the result of animmune response to an allergen in contact with the subject.
 79. A methodof inducing tolerance to a specific antigen in a subject in needthereof, wherein the subject's immune system is exposed to the specificantigen, the method comprising administering to the subject an effectiveamount of at least one cytokine selected from the group consisting ofIL-5, IL-12, IL-23 and IFN-γ, or a biologically active fragment thereof,or a functionally equivalent molecule thereof.
 80. A method of treatingor preventing in a subject in need thereof a disease resulting from animmune response to an antigen, the method comprising administering tothe subject an effective amount of at least one cytokine selected fromthe group consisting of IL-5, IL-12, IL-23 and IFN-γ, or a biologicallyactive fragment thereof, or a functionally equivalent molecule thereof.81. The method of claim 80, wherein the disease is: (a) a diseaseassociated with an immune response to an autoantigen; (b) a disease thatis the result of an immune response to a non-self antigen in contactwith the subject; or (c) a disease that is the result of an immuneresponse to an allergen in contact with the subject.
 82. A method ofinducing tolerance to a specific antigen in a subject in need thereof,wherein the subject's immune system is exposed to the specific antigen,the method comprising: (a) determining whether a sample of lymphocytescomprising CD4⁺,CD25⁺ T cells obtained from a subject comprisesCD4+,CD25+T cells that are responsive to one or more cytokines selectedfrom the group consisting of IL-5, IL-12, IL-23 and IFN-γ, or abiologically active fragment thereof, or a functionally equivalentmolecule thereof; and (b) administering an effective amount of one ormore cytokines selected from the group consisting of IL-5, IL-12, IL-23and IFN-γ, or a biologically active fragment thereof, or a functionallyequivalent molecule thereof, to which the CD4⁺,CD25⁺ T cells areresponsive.
 83. The method of claim 82 comprising, prior to orsimultaneously with, step (b), the step of administering to the subjectan effective amount of one or more cytokines selected from the groupconsisting of IL-2, a biologically active fragment thereof, or afunctionally equivalent molecule thereof, and IL-4, a biologicallyactive fragment thereof, or a functionally equivalent molecule thereof.84. A method of treating or preventing in a subject in need thereof adisease resulting from an immune response to an antigen, the methodcomprising: (a) determining whether a sample of lymphocytes comprisingCD4+,CD25+T cells obtained from a subject comprises CD4⁺,CD25⁺ T cellsthat are responsive to one or more cytokines selected from the groupconsisting of IL-5, IL-12, IL-23 and IFN-γ, or a biologically activefragment thereof, or a functionally equivalent molecule thereof; and (b)administering an effective amount of one or more cytokines selected fromthe group consisting of IL-5, IL-12, IL-23 and IFN-γ, or a biologicallyactive fragment thereof, or a functionally equivalent molecule thereof,to which the CD4⁺,CD25⁺ T cells are responsive.
 85. The method of claim84 comprising, prior to or simultaneously with, step (b), the step ofadministering to the subject an effective amount of one or morecytokines selected from the group consisting of IL-2, a biologicallyactive fragment thereof, or a functionally equivalent molecule thereof,and IL-4, a biologically active fragment thereof, or a functionallyequivalent molecule thereof.
 86. The method of claim 84, wherein thedisease is: (a) a disease associated with an immune response to anautoantigen; (b) a disease that is the result of an immune response to anon-self antigen in contact with the subject; or (c) a disease that isthe result of an immune response to an allergen in contact with thesubject.